Yield traits for maize

ABSTRACT

Methods for introgressing an allele of interest of a locus associated with a yield trait into  Zea mays  germplasm are provided. In some embodiments, the methods include providing a  Zea mays  plant that contains an allele of interest of a locus associated with a yield trait, wherein the locus associated with the yield trait is identifiable by PCR amplification of a  Zea mays  nucleic acid with a pair of oligonucleotides primers as disclosed herein, and introgressing the allele of interest into  Zea mays  germplasm that lacks the allele. Also provided are methods for identifying  Zea mays  plants that contain at least one allele associated with improved yield, improved maize plants, elite  Zea mays  plants, biomass produced from improved  Zea mays  plants, isolated and purified genetic markers, and compositions that include an amplification primer pair capable of amplifying a  Zea mays  nucleic acid to generate a  Zea mays  marker amplicon.

RELATED APPLICATION

The presently disclosed subject matter is a continuation of U.S. patentapplication Ser. No. 14/323,005 filed on Jul. 3, 2014, which is acontinuation of U.S. patent application Ser. No. 12/536,147 filed Aug.5, 2009 and granted on Aug. 19, 2014 as U.S. Pat. No. 8,809,621, whichis further incorporated by reference.

INCORPORATION OF THE SEQUENCE LISTING

A Sequence Listing is contained in the attached file named“72598-US-REG-C-NAT-1_SequenceListing_ST25”, which is 77 kilobytes andwas filed with U.S. patent application Ser. No. 12/536,147 filed Aug. 5,2009 for which this application claims the benefit thereof. The SequenceListing is attached and filed herewith and is incorporated herein byreference.

TECHNICAL FIELD

The presently disclosed subject matter relates to maize, such as maizeof the species Zea mays, and methods of breeding the same. Moreparticularly, the presently disclosed subject matter relates to maizelines, such as Zea mays lines, with one or more improved yield traits,and methods for breeding the same, which methods involve in someembodiments genetic marker analysis and/or nucleic acid sequenceanalysis.

BACKGROUND

A goal of plant breeding is to combine, in a single plant, variousdesirable traits. For field crops such as corn, these traits can includegreater yield and better agronomic quality. However, genetic loci thatinfluence yield and agronomic quality are not always known, and even ifknown, their contributions to such traits are frequently unclear. Thus,new loci that can positively influence such desirable traits need to beidentified and/or the abilities of known loci to do so need to bediscovered.

Once discovered, these desirable loci can be selected for as part of abreeding program in order to generate plants that carry desirabletraits. An exemplary embodiment of a method for generating such plantsincludes the transfer by introgression of nucleic acid sequences fromplants that have desirable genetic information into plants that do notby crossing the plants using traditional breeding techniques.

Desirable loci can be introgressed into commercially available plantvarieties using marker-assisted selection (MAS) or marker-assistedbreeding (MAB). MAS and MAB involves the use of one or more of themolecular markers for the identification and selection of those progenyplants that contain one or more loci that encode the desired traits.Such identification and selection can be based on selection ofinformative markers that are associated with desired traits. MAB canalso be used to develop near-isogenic lines (NIL) harboring loci ofinterest, allowing a more detailed study of the effect each locus canhave on a desired trait, and is also an effective method for developmentof backcross inbred line (BIL) populations.

What are needed, then, are new methods and compositions for geneticallyanalyzing Zea mays varieties and for employing the information obtainedfor producing new Zea mays plants that have improved traits.

SUMMARY

This summary lists several embodiments of the presently disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently disclosed subjectmatter, whether listed in this summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

The presently disclosed subject matter provides methods forintrogressing an allele of interest of a locus associated with a yieldtrait into Zea mays germplasm. In some embodiments, the methods comprise(a) selecting a Zea mays plant that comprises an allele of interest of alocus associated with a yield trait, wherein the locus associated withthe yield trait is identifiable by PCR amplification of a Zea maysnucleic acid with a pair of oligonucleotides primers selected from, butnot limited to, (i) primer pair 1 represented by a primer comprising SEQID NO: 2 and a primer comprising SEQ ID NO: 3; (ii) primer pair 2represented by a primer comprising SEQ ID NO: 5 and a primer comprisingSEQ ID NO: 6; (iii) primer pair 3 represented by a primer comprising SEQID NO: 8 and a primer comprising SEQ ID NO: 9; (iv) primer pair 4represented by a primer comprising SEQ ID NO: 11 and a primer comprisingSEQ ID NO: 12; (v) primer pair 5 represented by a primer comprising SEQID NO: 14 and a primer comprising SEQ ID NO: 15; (vi) primer pair 6represented by a primer comprising SEQ ID NO: 17 and a primer comprisingSEQ ID NO: 18; (vii) primer pair 7 represented by a primer comprisingSEQ ID NO: 20 and a primer comprising SEQ ID NO: 21; (viii) primer pair8 represented by a primer comprising SEQ ID NO: 23 and a primercomprising SEQ ID NO: 24; (ix) primer pair 9 represented by a primercomprising SEQ ID NO: 26 and a primer comprising SEQ ID NO: 27; (x)primer pair 10 represented by a primer comprising SEQ ID NO: 29 and aprimer comprising SEQ ID NO: 30; (xi) primer pair 11 represented by aprimer comprising SEQ ID NO: 32 and a primer comprising SEQ ID NO: 33;(xii) primer pair 12 represented by a primer comprising SEQ ID NO: 35and a primer comprising SEQ ID NO: 36; (xiii) primer pair 13 representedby a primer comprising SEQ ID NO: 38 and a primer comprising SEQ ID NO:39; (xiv) primer pair 14 represented by a primer comprising SEQ ID NO:41 and a primer comprising SEQ ID NO: 42; (xv) primer pair 15represented by a primer comprising SEQ ID NO: 44 and a primer comprisingSEQ ID NO: 45; (xvi) primer pair 16 represented by a primer comprisingSEQ ID NO: 47 and a primer comprising SEQ ID NO: 48; (xvii) primer pair17 represented by a primer comprising SEQ ID NO: 50 and a primercomprising SEQ ID NO: 51; (xviii) primer pair 18 represented by a primercomprising SEQ ID NO: 53 and a primer comprising SEQ ID NO: 54; (xix)primer pair 19 represented by a primer comprising SEQ ID NO: 56 and aprimer comprising SEQ ID NO: 57; (xx) primer pair 20 represented by aprimer comprising SEQ ID NO: 59 and a primer comprising SEQ ID NO: 60;(xxi) primer pair 21 represented by a primer comprising SEQ ID NO: 62and a primer comprising SEQ ID NO: 63; and (xxii) primer pair 22represented by a primer comprising SEQ ID NO: 65 and a primer comprisingSEQ ID NO: 66; and (b) introgressing the allele of interest into Zeamays germplasm that lacks the allele. In some embodiments, the allele ofinterest comprises a nucleotide sequence as set forth in any of SEQ IDNOs: 67-132. In some embodiments, the yield trait comprises a starchtrait, a protein trait, an oil trait, an ethanol production trait, or acombination thereof. In some embodiments, the allele of interest is afavorable allele that positively correlates with an improved starch-,oil-, and/or ethanol production-associated trait or that negativelycorrelates with an improved protein-associated trait.

The presently disclosed subject matter also provides methods foridentifying a Zea mays plant comprising at least one allele associatedwith improved yield. In some embodiments, the methods comprise (a)genotyping at least one Zea mays plant with at least one nucleic acidmarker selected from, but not limited to, SEQ ID NOs: 1, 4, 7, 10, 13,16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, and111-173; and (b) selecting at least one Zea mays plant comprising anallele of at least one of the at least one nucleic acid marker that isassociated with improved yield. In some embodiments, the alleleassociated with improved yield comprises a nucleotide sequence as setforth in any of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34,37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67-132. In some embodiments, theallele associated with improved yield is a favorable allele thatpositively correlates with an improved starch-, oil-, and/or ethanolproduction-associated trait or that negatively correlates with animproved protein-associated trait.

In some embodiments of the presently disclosed methods, the favorableallele comprises a nucleotide sequence comprising (i) an A at nucleotideposition 701 of SEQ ID NO: 1 or at nucleotide position 30 of SEQ ID NO:111; (ii) a G at nucleotide position 498 of SEQ ID NO: 4 or atnucleotide position 23 of SEQ ID NO: 112; (iii) a T at nucleotideposition 587 of SEQ ID NO: 7 or at nucleotide position 33 of SEQ ID NO:113; (iv) a G at nucleotide position 708 of SEQ ID NO: 10 or atnucleotide position 76 of SEQ ID NO: 114; (v) a C at nucleotide position140 of SEQ ID NO: 13 or at nucleotide position 58 of SEQ ID NO: 115;(vi) an A at nucleotide position 116 of SEQ ID NO: 16 or at nucleotideposition 33 of SEQ ID NO: 116; (vii) an A at nucleotide position 269 ofSEQ ID NO: 19 or at nucleotide position 32 of SEQ ID NO: 117; (viii) anA at nucleotide position 280 of SEQ ID NO: 22 or at nucleotide position23 of SEQ ID NO: 118; (ix) a T at nucleotide position 374 of SEQ ID NO:25 or at nucleotide position 46 of SEQ ID NO: 119; (x) a G at nucleotideposition 236 of SEQ ID NO: 28 or at nucleotide position 41 of SEQ ID NO:120; (xi) a G at nucleotide position 605 of SEQ ID NO: 31 or atnucleotide position 32 of SEQ ID NO: 121; (xii) a CGA trinucleotidesequence at nucleotide positions 349-351 of SEQ ID NO: 34 or atnucleotide positions 48-50 of SEQ ID NO: 122; (xiii) a C at nucleotideposition 389 of SEQ ID NO: 37 or at nucleotide position 45 of SEQ ID NO:123; (xiv) a G at nucleotide position 66 of SEQ ID NO: 40 or atnucleotide position 44 of SEQ ID NO: 124; (xv) a T at nucleotideposition 278 of SEQ ID NO: 43 or at nucleotide position 48 of SEQ ID NO:125; (xvi) a G at nucleotide position 463 of SEQ ID NO: 46 or atnucleotide position 20 of SEQ ID NO: 126; (xvii) a G at nucleotideposition 510 of SEQ ID NO: 49 or at nucleotide position 126 of SEQ IDNO: 127; (xviii) a G at nucleotide position 134 of SEQ ID NO: 52 or atnucleotide position 126 of SEQ ID NO: 128; (xix) an A at nucleotideposition 367 of SEQ ID NO: 55 or at nucleotide position 32 of SEQ ID NO:129; (xx) a G at nucleotide position 119 of SEQ ID NO: 58 or atnucleotide position 23 of SEQ ID NO: 130; (xxi) a G at nucleotideposition 347 of SEQ ID NO: 61 or at nucleotide position 53 of SEQ ID NO:131; or (xxii) and an A at nucleotide position 356 of SEQ ID NO: 64 orat nucleotide position 43 of SEQ ID NO: 132.

The presently disclosed subject matter also provides improved Zea maysplants produced by the presently disclosed methods, as well as parts,seeds, progeny, and tissue cultures thereof. In some embodiments, thepart, seed, progeny, or tissue culture thereof comprises at least oneallele of interest for each of at least two distinct loci associatedwith yield traits, and further wherein the improved plant or the part,seed, progeny, or tissue culture thereof comprises (a) a desired starchallele and a desired ethanol production allele; and/or (b) a desiredstarch allele and a desired protein allele. In some embodiments, theimproved Zea mays plant or the part, seed, progeny, or tissue culturethereof, comprises a desired allele for increased starch and a desiredallele for decreased protein.

The presently disclosed subject matter also provides elite Zea maysplants produced from the improved Zea mays plants disclosed herein.

The presently disclosed subject matter also provides biomass producedfrom the improved Zea mays plants disclosed herein, or from a progenyplant thereof, or from a part, seed, or tissue culture thereof.

The presently disclosed subject matter also provides isolated andpurified genetic markers associated with a yield trait in Zea mays. Insome embodiments, the isolated and purified genetic marker (i) comprisesa nucleotide sequence as set forth in any of SEQ ID NOs: 1-173, or thereverse complement thereof, or an informative fragment thereof; and/or(ii) comprises a nucleotide sequence of an amplification product or aninformative fragment thereof from a nucleic acid sample isolated from aZea mays plant, wherein the amplification product is produced byamplifying a Zea mays nucleic acid using a pair of oligonucleotideprimers selected from, but not limited to, SEQ ID NOs: 2 and 3; SEQ IDNOs: 5 and 6; SEQ ID SEQ ID NOs: 8 and 9; SEQ ID NOs: 11 and 12; SEQ IDNOs: 14 and 15; SEQ ID NOs: 17 and 18; SEQ ID NOs: 20 and 21; SEQ IDNOs: 23 and 24; SEQ ID NOs: 26 and 27; or SEQ ID NOs: 29 and 30; SEQ IDNOs: 32 and 33; SEQ ID NOs: 35 and 36; SEQ ID NOs: 38 and 39; SEQ IDNOs: 41 and 42; SEQ ID NOs: 44 and 45; SEQ ID NOs: 47 and 48; SEQ IDNOs: 50 and 51; SEQ ID NOs: 53 and 54; SEQ ID NOs: 56 and 57; SEQ IDNOs: 59 and 60; SEQ ID NOs: 62 and 63; and SEQ ID NOs: 65 and 66. Insome embodiments, the isolated and purified genetic marker permitsidentification of a nucleotide in the genome of a Zea mays plant thatcorresponds to the nucleotide present at any of nucleotide position 30of SEQ ID NO: 111; nucleotide position 23 of SEQ ID NO: 112; nucleotideposition 33 of SEQ ID NO: 113; nucleotide position 76 of SEQ ID NO: 114;nucleotide position 58 of SEQ ID NO: 115; nucleotide position 33 of SEQID NO: 116; nucleotide position 32 of SEQ ID NO: 117; nucleotideposition 23 of SEQ ID NO: 118; nucleotide position 46 of SEQ ID NO: 119;nucleotide position 41 of SEQ ID NO: 120; nucleotide position 32 of SEQID NO: 121; nucleotide positions 48-50 of SEQ ID NO: 122; nucleotideposition 45 of SEQ ID NO: 123; nucleotide position 44 of SEQ ID NO: 124;nucleotide position 48 of SEQ ID NO: 125; nucleotide position 20 of SEQID NO: 126; nucleotide position 126 of SEQ ID NO: 127; nucleotideposition 126 of SEQ ID NO: 128; nucleotide position 32 of SEQ ID NO:129; nucleotide position 23 of SEQ ID NO: 130; nucleotide position 53 ofSEQ ID NO: 131; and nucleotide position 43 of SEQ ID NO: 132. In someembodiments, the isolated and purified genetic marker further comprisesa detectable moiety.

The presently disclosed subject matter also provides compositionscomprising amplification primer pairs capable of amplifying a Zea maysnucleic acid to generate Zea mays marker amplicons. In some embodiments,the Zea mays marker amplicons correspond to any of SEQ ID NOs: 1, 4, 7,10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61,64, and 111-173.

Thus, it is an object of the presently disclosed subject matter toprovide methods for conveying one or more yield traits into maizegermplasm.

An object of the presently disclosed subject matter having been statedhereinabove, and which is achieved in whole or in part by the presentlydisclosed subject matter, other objects will become evident as thedescription proceeds when taken in connection with the accompanyingFigures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a scatter plot correlation of yield (e.g., starch, protein,oil, and/or ethanol production) traits from the inbred platform. Thenumbers in the top left corner of each box present pairwise correlationsbetween the traits (e.g., a positive correlation (r=0.2203) between oil(Oil_db) and protein (PRTNP) traits and a negative correlation(r=−0.7667) between protein (PRTNP) and digestibility at 72 hours (DGST72)).

FIG. 2 is a bar graph showing a random sampling of 97 lines that werepresent in the inbred panel and the inbred platform describedhereinbelow. For each bar, the number of occurrences represents thenumber of lines out of the 97 lines that had a genomic contribution thatcould be traced to the indicated line. SS: stiff stalk; NSS: non-stiffstalk.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 is a nucleotide sequence that is associated with the yieldloci MTL1, MTPL1, and MTSL1, a subsequence of which (SEQ ID NO: 111) canbe amplified from chromosome 5 of the Zea mays genome using thepolymerase chain reaction with amplification primers comprising thenucleotide sequences set forth in SEQ ID NOs: 2 and 3.

SEQ ID NO: 4 is a nucleotide sequence that is associated with the yieldloci MTL2 and MTSL2, a subsequence of which (SEQ ID NO: 112) can beamplified from chromosome 5 of the Zea mays genome using the polymerasechain reaction with amplification primers comprising the nucleotidesequences set forth in SEQ ID NOs: 5 and 6.

SEQ ID NO: 7 is a nucleotide sequence that is associated with the yieldloci MTL3 and MTSL3, a subsequence of which (SEQ ID NO: 113) can beamplified from chromosome 5 of the Zea mays genome using the polymerasechain reaction with amplification primers comprising the nucleotidesequences set forth in SEQ ID NOs: 8 and 9.

SEQ ID NO: 10 is a nucleotide sequence that is associated with the yieldloci MTL4, MTPL2, and MTSL4, a subsequence of which (SEQ ID NO: 114) canbe amplified from chromosome 2 of the Zea mays genome using thepolymerase chain reaction with amplification primers comprising thenucleotide sequences set forth in SEQ ID NOs: 11 and 12.

SEQ ID NO: 13 is a nucleotide sequence that is associated with the yieldlocus MTL5, a subsequence of which (SEQ ID NO: 115) can be amplifiedfrom chromosome 2 of the Zea mays genome using the polymerase chainreaction with amplification primers comprising the nucleotide sequencesset forth in SEQ ID NOs: 14 and 15.

SEQ ID NO: 16 is a nucleotide sequence that is associated with the yieldlocus MTL6, a subsequence of which (SEQ ID NO: 116) can be amplifiedfrom chromosome 5 of the Zea mays genome using the polymerase chainreaction with amplification primers comprising the nucleotide sequencesset forth in SEQ ID NOs: 17 and 18.

SEQ ID NO: 19 is a nucleotide sequence that is associated with the yieldloci MTL7 and MTPL3, a subsequence of which (SEQ ID NO: 117) can beamplified from chromosome 7 of the Zea mays genome using the polymerasechain reaction with amplification primers comprising the nucleotidesequences set forth in SEQ ID NOs: 20 and 21.

SEQ ID NO: 22 is a nucleotide sequence that is associated with the yieldloci MTL8 and MTPL4, a subsequence of which (SEQ ID NO: 118) can beamplified from chromosome 5 of the Zea mays genome using the polymerasechain reaction with amplification primers comprising the nucleotidesequences set forth in SEQ ID NOs: 23 and 24.

SEQ ID NO: 25 is a nucleotide sequence that is associated with the yieldlocus MTL9, a subsequence of which (SEQ ID NO: 119) can be amplifiedfrom chromosome 5 of the Zea mays genome using the polymerase chainreaction with amplification primers comprising the nucleotide sequencesset forth in SEQ ID NOs: 26 and 27.

SEQ ID NO: 28 is a nucleotide sequence that is associated with the yieldloci MTL10 and MTOL3, a subsequence of which (SEQ ID NO: 120) can beamplified from chromosome 1 of the Zea mays genome using the polymerasechain reaction with amplification primers comprising the nucleotidesequences set forth in SEQ ID NOs: 29 and 30.

SEQ ID NO: 31 is a nucleotide sequence that is associated with the yieldlocus MTL11, a subsequence of which (SEQ ID NO: 121) can be amplifiedfrom chromosome 2 of the Zea mays genome using the polymerase chainreaction with amplification primers comprising the nucleotide sequencesset forth in SEQ ID NOs: 32 and 33.

SEQ ID NO: 34 is a nucleotide sequence that is associated with the yieldlocus MTL12, a subsequence of which (SEQ ID NO: 122) can be amplifiedfrom chromosome 10 of the Zea mays genome using the polymerase chainreaction with amplification primers comprising the nucleotide sequencesset forth in SEQ ID NOs: 35 and 36.

SEQ ID NO: 37 is a nucleotide sequence that is associated with the yieldloci MTL13 and MTSL5, a subsequence of which (SEQ ID NO: 123) can beamplified from chromosome 8 of the Zea mays genome using the polymerasechain reaction with amplification primers comprising the nucleotidesequences set forth in SEQ ID NOs: 38 and 39.

SEQ ID NO: 40 is a nucleotide sequence that is associated with the yieldloci MTL14 and MTPL9, a subsequence of which (SEQ ID NO: 124) can beamplified from chromosome 1 of the Zea mays genome using the polymerasechain reaction with amplification primers comprising the nucleotidesequences set forth in SEQ ID NOs: 41 and 42.

SEQ ID NO: 43 is a nucleotide sequence that is associated with the yieldlocus MTL15, a subsequence of which (SEQ ID NO: 125) can be amplifiedfrom chromosome 5 of the Zea mays genome using the polymerase chainreaction with amplification primers comprising the nucleotide sequencesset forth in SEQ ID NOs: 44 and 45.

SEQ ID NO: 46 is a nucleotide sequence that is associated with the yieldlocus MTL16, a subsequence of which (SEQ ID NO: 126) can be amplifiedfrom chromosome 1 of the Zea mays genome using the polymerase chainreaction with amplification primers comprising the nucleotide sequencesset forth in SEQ ID NOs: 47 and 48.

SEQ ID NO: 49 is a nucleotide sequence that is associated with the yieldloci MTL17 and MTPL8, a subsequence of which (SEQ ID NO: 127) can beamplified from chromosome 5 of the Zea mays genome using the polymerasechain reaction with amplification primers comprising the nucleotidesequences set forth in SEQ ID NOs: 50 and 51.

SEQ ID NO: 52 is a nucleotide sequence that is associated with the yieldloci MTL18 and MTPL7, a subsequence of which (SEQ ID NO: 128) can beamplified from chromosome 10 of the Zea mays genome using the polymerasechain reaction with amplification primers comprising the nucleotidesequences set forth in SEQ ID NOs: 53 and 54.

SEQ ID NO: 55 is a nucleotide sequence that is associated with the yieldlocus MTOL1, a subsequence of which (SEQ ID NO: 129) can be amplifiedfrom chromosome 1 of the Zea mays genome using the polymerase chainreaction with amplification primers comprising the nucleotide sequencesset forth in SEQ ID NOs: 56 and 57.

SEQ ID NO: 58 is a nucleotide sequence that is associated with the yieldlocus MTPL5, a subsequence of which (SEQ ID NO: 130) can be amplifiedfrom chromosome 4 of the Zea mays genome using the polymerase chainreaction with amplification primers comprising the nucleotide sequencesset forth in SEQ ID NOs: 59 and 60.

SEQ ID NO: 61 is a nucleotide sequence that is associated with the yieldloci MTSL6 and MTPL10, a subsequence of which (SEQ ID NO: 131) can beamplified from chromosome 6 of the Zea mays genome using the polymerasechain reaction with amplification primers comprising the nucleotidesequences set forth in SEQ ID NOs: 62 and 63.

SEQ ID NO: 64 is a nucleotide sequence that is associated with the yieldloci MTPL6 and MTOL2, a subsequence of which (SEQ ID NO: 132) can beamplified from chromosome 7 of the Zea mays genome using the polymerasechain reaction with amplification primers comprising the nucleotidesequences set forth in SEQ ID NOs: 65 and 66.

SEQ ID NOs: 67-110 are nucleotide sequences of oligonucleotide probesassociated with MTL 1-18, MTPL 1-9, MTSL 1-6, and MTOL 1-3 that can beemployed to distinguish between alternative alleles of these loci as setforth in Table 1 below.

TABLE 1 SEQ ID NOs. for Assay Oligonucleotides Locus Nucleotide AlleleDetected: Assayed Assayed Primer SEQ ID NO. MTL1/MTPL1/MTSL1 701 A: SEQID NO: 67 (SEQ ID NO: 1) G: SEQ ID NO: 68 MTL2/MTSL2 498 G: SEQ ID NO:69 (SEQ ID NO: 4) A: SEQ ID NO: 70 MTL3/MTSL3 587 T: SEQ ID NO: 71 (SEQID NO: 7) C: SEQ ID NO: 72 MTL4/MTPL2/MTSL4 708 G: SEQ ID NO: 73 (SEQ IDNO: 10) A: SEQ ID NO: 74 MTL5 140 C: SEQ ID NO: 75 (SEQ ID NO: 13) T:SEQ ID NO: 76 MTL6 116 A: SEQ ID NO: 77 (SEQ ID NO: 16) G: SEQ ID NO: 78MTL7/MTPL3 269 A: SEQ ID NO: 79 (SEQ ID NO: 19) G: SEQ ID NO: 80MTL8/MTPL4 280 A: SEQ ID NO: 81 (SEQ ID NO: 22) G: SEQ ID NO: 82 MTL9374 T: SEQ ID NO: 83 (SEQ ID NO: 25) G: SEQ ID NO: 84 MTL10/MTOL3 236 G:SEQ ID NO: 85 (SEQ ID NO: 28) A: SEQ ID NO: 86 MTL11 605 G: SEQ ID NO:87 (SEQ ID NO: 31) A: SEQ ID NO: 88 MTL12 349 C: SEQ ID NO: 89 (SEQ IDNO: 34) T: SEQ ID NO: 90 MTL13/MTSL5 389 C: SEQ ID NO: 91 (SEQ ID NO:37) T: SEQ ID NO: 92 MTL14/MTPL9 66 G: SEQ ID NO: 93 (SEQ ID NO: 40) A:SEQ ID NO: 94 MTL15 278 T: SEQ ID NO: 95 (SEQ ID NO: 43) C: SEQ ID NO:96 MTL16 463 G: SEQ ID NO: 97 (SEQ ID NO: 46) A: SEQ ID NO: 98MTL17/MTPL8 510 G: SEQ ID NO: 99 (SEQ ID NO: 49) A: SEQ ID NO: 100MTL18/MTPL7 134 G: SEQ ID NO: 101 (SEQ ID NO: 52) T: SEQ ID NO: 102MTOL1 367 A: SEQ ID NO: 103 (SEQ ID NO: 55) G: SEQ ID NO: 104 MTPL5 119G: SEQ ID NO: 105 (SEQ ID NO: 58) C: SEQ ID NO: 106 MTSL6/MTPL10 347 G:SEQ ID NO: 107 (SEQ ID NO: 61) T: SEQ ID NO: 108 MTPL6/MTOL2 356 A: SEQID NO: 109 (SEQ ID NO: 64) G: SEQ ID NO: 110

SEQ ID NOs: 111-132 are nucleotide sequences associated with MTL 1-18,MTPL 1-9, MTSL 1-6, and/or MTOL 1-3 that can be amplified from Zea maysnucleic acids using sets of oligonucleotide primers as set forth inTable 2 below.

TABLE 2 SEQ ID NOs. for Zea mays Sequences that can be Amplified by PCRPrimers and Resultant Amplicon Sizes SEQ Exemplary AmplificationAmplicon ID NO: Primer SEQ ID NOs: Size (bp) 111 2 and 3 106 112 5 and 6136 113 8 and 9 160 114 11 and 12 122 115 14 and 15 120 or 121 116 17and 18 84 117 20 and 21 74 118 23 and 24 46 119 26 and 27 74 120 29 and30 87 121 32 and 33 86 122 35 and 34 141 or 144 123 38 and 39 101 124 41and 42 107 125 44 and 45 227 126 47 and 48 67 127 50 and 51 146 128 53and 54 153 129 56 and 57 66 130 59 and 60 59 131 62 and 63 107 132 65and 66 117

SEQ ID NOs. 133-152 are Zea mays genomic DNA sequences present in theGENBANK® database that correspond to the nucleotide sequences of SEQ IDNOs: 1-124 and 126-132 as set forth in Table 3 below. Subsequences ofthese sequences can also be amplified using primer pairs to yieldamplicons as also set forth in Table 3.

TABLE 3 GENBANK ® Database Sequences that Correspond to SEQ ID NOs:1-124 and 126-132 Exemplary Amplifica- SEQ GENBANK ® tion PrimerAmplicon Amplicon ID Accession Nucleotide SEQ SEQ Size NO: No. PositionsID NOs: ID NO. (bp) 133 AC209208.3 49,337 to 2 and 3 153 106 50,164 134AC206616.3 43,103 to 5 and 6 154 133 43,821 134 AC206616.3 43,103 to 8and 9 155 156 43,821 135 AC204769.3 50,033 to 11 and 12 156 121 50,828136 AC214243.4 99,595 to 14 and 15 157 120 100,453 137 AC185458.4 56,729to 17 and 18 158 84 57,382 138 AC211735.3 113,858 to 20 and 21 159 74114,950 139 AC212758.3 121,592 to 23 and 24 160 46 123,221 140AC203779.6 159,378 to 26 and 27 161 74 160,234 141 AC196146.3 4367 to 29and 30 162 87 4883 142 AC214129.2 106,354 to 32 and 33 163 86 107,115143 AC183312.5 2608 to 35 and 34 164 131 3395 144 AC194834.3 116,067 to38 and 39 165 101 116,863 145 AC197472.2 66,678 to 41 and 42 166 10767,232 146 AC197469.3 173,922 to 47 and 48 167 67 174,662 147 AC210263.4181,778 to 50 and 51 168 146 182,591 148 AC203332.3 120,187 to 53 and 54169 150 121,112 149 AC198211.4 85,472 to 56 and 57 170 66 86,260 150AC191759.3 147,402 to 59 and 60 171 59 148,055 151 AC204581.3 166,218 to62 and 63 172 101 166,660 152 AC212580.4 100,621 to 65 and 66 173 117101,358

SEQ ID NOs: 133-152 have been added to the GENBANK® database by theGenome Sequencing Center, Washington University School of Medicine, St.Louis, Mo., United States of America. As set forth in the annotations tothese database entries, the sequences were part of an effort by TheMaize Sequencing Consortium to sequence the genome of Zea mays.Currently, the sequencing effort has not been completed, and variousportions of the Zea mays genome remain unsequenced.

As can be seen in the above Tables, certain of the sequences of SEQ IDNOs: 1-173 are related to each other. By way of example, SEQ ID NO: 1 isa nucleotide sequence from Zea mays. A subsequence of SEQ ID NO: 1 canbe amplified in an amplification reaction (e.g., a PCR) usingoligonucleotide primers having the sequences set forth in SEQ ID NOs: 2and 3 to yield an amplicon that in some embodiments has a nucleotidesequence as set forth in SEQ ID NO: 111, which has a size of 106basepairs (bp). At position 701 of SEQ ID NO: 1 there is an SNP, and thespecific nucleotide that is present in any nucleic acid sample at thisposition can be determined using oligonucleotides that have thesequences set forth in SEQ ID NOs: 67 and 68.

Additionally, GENBANK® Accession No. AC209208.3 includes a subsequence(i.e., nucleotides 49,337 to 50,164; SEQ ID NO: 133) that itself (or itsreverse complement) is highly similar to SEQ ID NO: 1 and thus ispresent at the same locus from which SEQ ID NO: 1 is derived. Thedifferences between the two sequences (which can be identified using aBLAST® algorithm, a ClustalX algorithm, or any other appropriate methodof analysis) can be attributable to normal variation within Zea mayspopulations. A subsequence of SEQ ID NO: 133 can also be amplified in anamplification reaction (e.g., a PCR) using oligonucleotide primershaving the sequences set forth in SEQ ID NOs: 2 and 3 to yield anamplicon which in some embodiments has a nucleotide sequence as setforth in SEQ ID NO: 153, which has a size of 106 basepairs (bp).Oligonucleotides with the sequences set forth in SEQ ID NOs: 67 and 68can also be used to assay the base that is present at the position thatcorresponds to position 701 of SEQ ID NO: 1, which in this case is alsoposition 701 of SEQ ID NO: 133 and is position 30 of SEQ ID NO: 153.

For SEQ ID NOs: 134-173, similar interrelationships exist with SEQ IDNOs: 4-42, 46-66, 69-110, and 112-132 as are described hereinabove, andwould be identifiable by one of ordinary skill in the art using routinesequence analysis techniques. It is noted that with respect to SEQ IDNOs: 43-45, the complete nucleotide sequence of a genomic clone thatincludes the full length sequence that corresponds to these sequences(as well as to SEQ ID NO: 125) has not been yet been added to theGENBANK® database by The Maize Sequencing Consortium. As such, asequence from the GENBANK® database that can be amplified using primerscomprising SEQ ID NOs: 44 and 45 is not included in the Tables above. Itis further noted, however, that nucleotides 10,855-10,962 and103,384-103,629 of GENBANK® Accession No. AC204604.3 correspond tosubsequences of SEQ ID NOs: 43 and 125.

DETAILED DESCRIPTION I. Definitions

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

All technical and scientific terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. References to techniques employedherein are intended to refer to the techniques as commonly understood inthe art, including variations on those techniques or substitutions ofequivalent techniques that would be apparent to one of skill in the art.While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. For example, the phrase “a marker” refers to one or moremarkers. Similarly, the phrase “at least one”, when employed herein torefer to an entity, refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more of that entity,including but not limited to whole number values between 1 and 100 andgreater than 100.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about”. The term “about”, as used herein when referring to ameasurable value such as an amount of mass, weight, time, volume,concentration or percentage is meant to encompass variations of in someembodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, insome embodiments ±1%, in some embodiments ±0.5%, and in some embodiments±0.1% from the specified amount, as such variations are appropriate toperform the disclosed methods. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in this specification andattached claims are approximations that can vary depending upon thedesired properties sought to be obtained by the presently disclosedsubject matter.

As used herein, the term “allele” refers to a variant or an alternativesequence form at a genetic locus; in diploids a single allele at eachlocus is inherited by a progeny individual separately from each parent.The term “allele” thus refers to any of one or more alternative forms ofa nucleotide sequence of a gene, all of which relate to at least onetrait or characteristic, in a diploid cell. Two alleles of a given geneoccupy corresponding loci on a pair of homologous chromosomes, althoughone of ordinary skill in the art understands that the alleles in anyparticular individual do not necessarily represent all of the allelesthat are present in the species. Since the presently disclosed subjectmatter relates in some embodiments to MTLs (i.e., genomic regions thatcan comprise one or more genes or regulatory sequences), it is in someinstances more accurate to refer to a “haplotype” (i.e., an allele of achromosomal segment) instead of “allele”. However, in such instances,the term “allele” should be understood to comprise the term “haplotype”.

As used herein, the phrase “associated with” refers to a recognizableand/or assayable relationship between two entities. For example, thephrase “associated with an ethanol production trait” refers to a trait,locus, MTL gene, allele, marker, phenotype, etc., or the expressionthereof, the presence or absence of which can influence an extent,degree, and/or rate at which a plant that has the ethanol productiontrait produces ethanol. Similarly, the phrase “associated with a starchtrait” refers to a trait, locus, MTL gene, allele, marker, phenotype,etc., or the expression thereof, the presence or absence of which caninfluence the production of starch in a plant.

As used herein, the term “backcross”, and grammatical variants thereof,refers to a process in which a breeder crosses a progeny individual backto one of its parents: for example, a first generation F1 with one ofthe parental genotypes of the F1 individual. In some embodiments, abackcross is performed repeatedly, with a progeny individual of eachsuccessive backcross generation being itself backcrossed to the sameparental genotype.

As used herein, the term “chromosome” is used in its art-recognizedmeaning of the self-replicating genetic structure in the cellularnucleus containing the cellular DNA and bearing in its nucleotidesequence the linear array of genes. The Zea mays chromosome numbersdisclosed herein refer to those as set forth in Perin et al., 2002,which relates to a reference nomenclature system adopted by L'institutNational da la Recherché Agronomique (INRA; Paris, France).

As used herein, the phrase “consensus sequence” refers to a sequence ofDNA built to identify nucleotide differences (e.g., SNP and Indelpolymorphisms) in alleles at a locus. A consensus sequence can be eitherstrand of DNA at the locus and states the nucleotide(s) at one or morepositions (e.g., at one or more SNPs and/or at one or more Indels) inthe locus. In some embodiments, a consensus sequence is used to designprimers and probes for detecting polymorphisms in the locus.

As used herein, the terms “cultivar” and “variety” refer to a group ofsimilar plants that by structural or genetic features and/or performancecan be distinguished from other varieties within the same species.

As used herein, the phrase “elite line” refers to any line that issubstantially homozygous and has resulted from breeding and selectionfor superior agronomic performance.

As used herein, the term “gene” refers to a hereditary unit including asequence of DNA that occupies a specific location on a chromosome andthat contains the genetic instruction for a particular characteristicsor trait in an organism.

As used herein, the phrase “genetic map” refers to the ordered list ofloci usually relevant to position on a chromosome.

As used herein, the phrase “genetic marker” refers to a nucleic acidsequence (e.g., a polymorphic nucleic acid sequence) that has beenidentified as associated with a locus or allele of interest and that isindicative of the presence or absence of the locus or allele of interestin a cell or organism. Examples of genetic markers include, but are notlimited to genes, DNA or RNA-derived sequences, promoters, anyuntranslated regions of a gene, microRNAs, siRNAs, QTLs, transgenes,mRNAs, ds RNAs, transcriptional profiles, and methylation patterns.

As used herein, the term “genotype” refers to the genetic component of aphenotype of interest, a plurality of phenotypes of interest, or anentire cell or organism. Genotypes can be indirectly characterized usingmarkers and/or directly characterized by nucleic acid sequencing.

As used herein, the term “heterozygous” refers to a genetic conditionthat exists in a cell or an organism when different alleles reside atcorresponding loci on homologous chromosomes. As used herein, the term“homozygous” refers to a genetic condition existing when identicalalleles reside at corresponding loci on homologous chromosomes. It isnoted that both of these terms can refer to single nucleotide positions,multiple nucleotide positions, whether contiguous or not, or entire locion homologous chromosomes.

As used herein, the term “hybrid” refers to a seed and the plant theseed develops into that result from crossing at least two geneticallydifferent plant parents.

As used herein, the term “hybrid” when used in the context of nucleicacids, refers to a double-stranded nucleic acid molecule, or duplex,formed by hydrogen bonding between complementary nucleotide bases. Theterms “hybridize” and “anneal” refer to the process by which singlestrands of nucleic acid sequences form double-helical segments throughhydrogen bonding between complementary bases.

As used herein, the phrase “ILLUMINA® GOLDENGATE® Assay” refers to ahigh throughput genotyping assay sold by Illumina Inc. of San Diego,Calif., United States of America that can generate SNP-specific PCRproducts. This assay is described in detail at the website of IlluminaInc. and in Fan et al., 2006.

As used herein, the term “improved”, and grammatical variants thereof,refers to a plant or a part, progeny, or tissue culture thereof, that asa consequence of having (or lacking) a particular yield associatedallele (such as, but not limited to those yield associated allelesdisclosed herein) is characterized by a higher or lower content of ayield associated trait, depending on whether the higher or lower contentis desired for a particular purpose.

As used herein, the term “inbred” refers to a substantially homozygousindividual or line. It is noted that the term can refer to individualsor lines that are substantially homozygous throughout their entiregenomes or that are substantially homozygous with respect tosubsequences of their genomes that are of particular interest.

As used herein, the phrase “immediately adjacent”, when used to describea nucleic acid molecule that hybridizes to DNA containing apolymorphism, refers to a nucleic acid that hybridizes to a DNA sequencethat directly abuts the polymorphic nucleotide base position. Forexample, a nucleic acid molecule that can be used in a single baseextension assay is “immediately adjacent” to the polymorphism.

As used herein, the phrase “interrogation position” refers to a physicalposition on a solid support that can be queried to obtain genotypingdata for one or more predetermined genomic polymorphisms.

As used herein, the terms “introgression”, “introgressed”, and“introgressing” refer to both a natural and artificial process wherebygenomic regions of one species, variety, or cultivar are moved into thegenome of another species, variety, or cultivar by crossing thosespecies. Exemplary methods for introgressing a trait of interestinclude, but are not limited to breeding an individual that has thetrait of interest to an individual that does not, and backcrossing anindividual that has the trait of interest to a recurrent parent.

As used herein, the term “linkage” refers to a phenomenon whereinalleles on the same chromosome tend to be transmitted together moreoften than expected by chance if their transmission were independent.Thus, two alleles on the same chromosome are said to be “linked” whenthey segregate from each other in the next generation in someembodiments less than 50% of the time, in some embodiments less than 25%of the time, in some embodiments less than 20% of the time, in someembodiments less than 15% of the time, in some embodiments less than 10%of the time, in some embodiments less than 9% of the time, in someembodiments less than 8% of the time, in some embodiments less than 7%of the time, in some embodiments less than 6% of the time, in someembodiments less than 5% of the time, in some embodiments less than 4%of the time, in some embodiments less than 3% of the time, in someembodiments less than 2% of the time, and in some embodiments less than1% of the time.

As such, “linkage” typically implies physical proximity on a chromosome.Thus, two loci are linked if they are within in some embodiments 20centiMorgans (cM), in some embodiments 15 cM, in some embodiments 12 cM,in some embodiments 10 cM, in some embodiments 9 cM, in some embodiments8 cM, in some embodiments 7 cM, in some embodiments 6 cM, in someembodiments 5 cM, in some embodiments 4 cM, in some embodiments 3 cM, insome embodiments 2 cM, and in some embodiments 1 cM of each other.Similarly, a Marker Trait Locus (MTL) of the presently disclosed subjectmatter is linked to a marker if it is in some embodiments within 20, 15,12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cM of the marker.

As used herein, the phrase “linkage group” refers to all of the genes orgenetic traits that are located on the same chromosome. Within thelinkage group, those loci that are close enough together can exhibitlinkage in genetic crosses. Since the probability of crossover increaseswith the physical distance between loci on a chromosome, loci for whichthe locations are far removed from each other within a linkage groupmight not exhibit any detectable linkage in direct genetic tests. Theterm “linkage group” is mostly used to refer to genetic loci thatexhibit linked behavior in genetic systems where chromosomal assignmentshave not yet been made. Thus, in the present context, the term “linkagegroup” is synonymous with the physical entity of a chromosome, althoughone of ordinary skill in the art will understand that a linkage groupcan also be defined as corresponding to a region of (i.e., less than theentirety) of a given chromosome.

As used herein, the phrase “linkage disequilibrium” is defined as changefrom the expected relative frequency of gamete types in a population ofmany individuals in a single generation such that two or more loci actas genetically linked loci. If the frequency in a population of allele Sis x, s is x′, B is y, and b is y′, then the expected frequency ofgenotype SB is xy, that of Sb is xy′, that of sB is x′y, and that of sbis x′y′, and any deviation from these frequencies is an example ofdisequilibrium.

As used herein, the term “locus” refers to an established position on achromosome of a species, and which may encompass a single nucleotide,several nucleotides, or more in a genomic region.

As used herein, the term “maize” refers to a plant, or a part thereof,of the species Zea mays, also referred to herein as Zea mays L.

As used herein, the phrase “maize-specific DNA sequence” refers to apolynucleotide sequence having a nucleotide sequence identity of in someembodiments more than 50%, in some embodiments more than 55%, in someembodiments more than 60%, in some embodiments more than 65%, in someembodiments more than 70%, in some embodiments more than 75%, in someembodiments more than 80%, in some embodiments more than 85%, in someembodiments more than 90%, in some embodiments more than 92%, in someembodiments more than 95%, in some embodiments more than 96%, in someembodiments more than 97%, in some embodiments more than 98%, and insome embodiments more than 99% with a sequence of the genome of thespecies Zea mays that shows the greatest similarity to it. In someembodiments and in the case of markers for any of MTL1-MTL18, MTPL1-10,MTSL1-6, and MTOL1-3 of the presently disclosed subject matter, amaize-specific DNA sequence comprises a part of a genomic DNA sequenceof a Zea mays plant that flanks one of the MTL1-MTL18, MTPL1-10,MTSL1-6, and/or MTOL1-3 loci.

As used herein, the terms “marker” and “molecular marker” are usedinterchangeably to refer to an identifiable position on a chromosome theinheritance of which can be monitored and/or a reagent that is used inmethods for visualizing differences in nucleic acid sequences present atsuch identifiable positions on chromosomes. Thus, in some embodiments amarker comprises a known or detectable nucleic acid sequence. Examplesof markers include, but are not limited to genetic markers, proteincomposition, protein levels, oil composition, oil levels, carbohydratecomposition, carbohydrate levels, fatty acid composition, fatty acidlevels, amino acid composition, amino acid levels, biopolymers, starchcomposition, starch levels, fermentable starch, fermentation yield,fermentation efficiency (e.g., captured as digestibility at 24, 48,and/or 72 hours), energy yield, secondary compounds, metabolites,morphological characteristics, and agronomic characteristics. DNA-basedmarkers include, but are not limited to restriction fragment lengthpolymorphisms (RFLPs), random amplified polymorphic DNA (RAPD),amplified fragment length polymorphisms (AFLPs), single strandconformation polymorphism (SSCPs), single nucleotide polymorphisms(SNPs), insertion/deletion mutations (Indels), simple sequence repeats(SSRs), microsatellite repeats, sequence-characterized amplified regions(SCARs), cleaved amplified polymorphic sequence (CAPS) markers, andisozyme markers, microarray-based technologies, TAQMAN® markers,ILLUMINA® GOLDENGATE® Assay markers, nucleic acid sequences, orcombinations of the markers described herein, which define a specificgenetic and chromosomal location. The phrase a “molecular marker linkedto an MTL” as defined herein can thus refer in some embodiments to SNPs,Indels, AFLP markers, or any other type of marker used in the field.

In some embodiments, a marker corresponds to an amplification productgenerated by amplifying a Zea mays nucleic acid with two oligonucleotideprimers, for example, by the polymerase chain reaction (PCR). As usedherein, the phrase “corresponds to an amplification product” in thecontext of a marker refers to a marker that has a nucleotide sequencethat is the same (allowing for mutations introduced by the amplificationreaction itself and/or naturally occurring and/or artificial alleleicdifferences) as an amplification product that is generated by amplifyingZea mays genomic DNA with a particular set of primers. In someembodiments, the amplifying is by PCR, and the primers are PCR primersthat are designed to hybridize to opposite strands of the Zea maysgenomic DNA in order to amplify a Zea mays genomic DNA sequence presentbetween the sequences to which the PCR primers hybridize in the Zea maysgenomic DNA. The amplified fragment that results from one or more roundsof amplification using such an arrangement of primers is a doublestranded nucleic acid, one strand of which has a nucleotide sequencethat comprises, in 5′ to 3′ order, the sequence of one of the primers,the sequence of the Zea mays genomic DNA located between the primers,and the reverse-complement of the second primer. Typically, the“forward” primer is assigned to be the primer that has the same sequenceas a subsequence of the (arbitrarily assigned) “top” strand of adouble-stranded nucleic acid to be amplified, such that the “top” strandof the amplified fragment includes a nucleotide sequence that is, in 5′to 3′ direction, equal to the sequence of the forward primer—thesequence located between the forward and reverse primers of the topstrand of the genomic fragment—the reverse-complement of the reverseprimer. Accordingly, a marker that “corresponds to” an amplifiedfragment is a marker that has the same sequence of one of the strands ofthe amplified fragment.

As used herein, the phrase “marker assay” refers to a method fordetecting a polymorphism at a particular locus using a particular methodsuch as but not limited to measurement of at least one phenotype (suchas seed color, oil content, or a visually detectable trait), restrictionfragment length polymorphism (RFLP), single base extension,electrophoresis, sequence alignment, allelic specific oligonucleotidehybridization (ASO), random amplified polymorphic DNA (RAPD),microarray-based technologies, TAQMAN® Assays, ILLUMINA® GOLDENGATE®Assay analysis, nucleic acid sequencing technologies, or any othertechnique that can be employed to identify the nucleotide sequence of anucleic acid.

As used herein, the terms “MTL1”, “MTL2”, “MTL3”, “MTL4”, “MTL5”,“MTL6”, “MTL7”, “MTL8”, “MTL9”, “MTL10”, “MTL11”, “MTL12”, “MTL13”,“MTL14”, “MTL15”, “MTL16”, “MTL17”, “MTL4”, and “MTL18” refer to genomicregions linked to ethanol production traits (alternatively referred toherein as “digestibility” traits), The terms “MTPL1”, “MTPL2”, “MTPL3”,“MTPL4”, “MTPL5”, “MTPL6”, “MTPL7”, “MTPL8”, “MTPL9”, and MTPL10 referto genomic regions linked to protein traits; “MTSL1”, “MTSL2”, “MTSL3”,“MTSL4”, “MTSL5”, and “MTSL6” refer to genomic regions linked to starchtraits; and “MTOL1”, “MTOL2”, and “MTOL3”, refer to genomic regionslinked oil traits as defined by markers present on Zea mays chromosomesand as described in more detail herein.

As used herein, the phrase “native trait” refers to any existingmonogenic or oligogenic trait in a certain crop's germplasm. Whenidentified through molecular marker(s), the information obtained can beused for the improvement of germplasm through marker assisted breedingof MTLs or genes.

As used herein, the phrases “nucleotide sequence identity” refers to thepresence of identical nucleotides at corresponding positions of twopolynucleotides. Polynucleotides have “identical” sequences if thesequence of nucleotides in the two polynucleotides is the same whenaligned for maximum correspondence. Sequence comparison between two ormore polynucleotides is generally performed by comparing portions of thetwo sequences over a comparison window to identify and compare localregions of sequence similarity, The comparison window is generally fromabout 20 to 200 contiguous nucleotides. The “percentage of sequenceidentity” for polynucleotides, such as 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 98, 99 or 100 percent sequence identity, can be determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide sequence in the comparisonwindow can include additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby: (a) determining the number of positions at which the identicalnucleic acid base occurs in both sequences to yield the number ofmatched positions; (b) dividing the number of matched positions by thetotal number of positions in the window of comparison; and (c)multiplying the result by 100 to yield the percentage of sequenceidentity. Optimal alignment of sequences for comparison can also beconducted by computerized implementations of known algorithms, or byvisual inspection. Readily available sequence comparison and multiplesequence alignment algorithms are, respectively, the Basic LocalAlignment Search Tool (BLAST®; Altschul et al., 1990; Altschul et al.,1997) and ClustalW programs, both available on the internet. Othersuitable programs include, but are not limited to, GAP, BestFit, PlotSimilarity, and FASTA, which are part of the Accelrys GCG Packageavailable from Accelrys, Inc. of San Diego, Calif., United States ofAmerica. In some embodiments, a percentage of sequence identity refersto sequence identity over the full length of one of the sequences beingcompared. In some embodiments, a calculation to determine a percentageof sequence identity does not include in the calculation any nucleotidepositions in which either of the compared nucleic acids includes an “n”(i.e., where any nucleotide could be present at that position).

As used herein, the phrases “progeny plant” refers to any plantresulting as progeny from a vegetative or sexual reproduction from oneor more parent plants or descendants thereof. For instance, a progenyplant can be obtained by cloning or selfing of a parent plant or bycrossing two parental plants and include selfings as well as the F1 orF2 or still further generations. An F1 is a first-generation progenyproduced from parents at least one of which is used for the first timeas donor of a trait, while progeny of second generation (F2) orsubsequent generations (F3, F4, and the like) are specimens producedfrom selfings, intercrosses, backcrosses, or other crosses of F1s, F2s,and the like. An F1 can thus be (and in some embodiments is) a hybridresulting from a cross between two true breeding parents (i.e., parentsthat are true-breeding are each homozygous for a trait of interest or anallele thereof), while an F2 can be (and in some embodiments is) aprogeny resulting from self-pollination of the F1 hybrids.

As used herein, the term “phenotype” refers to the detectablecharacteristics of a cell or organism due to genetics. Non-limitingexamples include protein content, starch content, oil content, anddigestibility (i.e., ethanol production) phenotypes, all of which areexemplary yield phenotypes.

As used herein, the phrase “phenotypic marker” refers to a marker thatcan be used to discriminate between different phenotypes.

As used herein, the term “plant” refers to an entire plant, its organs(i.e., leaves, stems, roots, flowers etc.), seeds, plant cells, andprogeny of the same. The term “plant cell” includes without limitationcells within seeds, suspension cultures, embryos, meristematic regions,callus tissue, leaves, shoots, gametophytes, sporophytes, pollen, andmicrospores. The phrase “plant part” refers to a part of a plant,including single cells and cell tissues such as plant cells that areintact in plants, cell clumps, and tissue cultures from which plants canbe regenerated. Examples of plant parts include, but are not limited to,single cells and tissues from pollen, ovules, leaves, embryos, roots,root tips, anthers, flowers, fruits, stems, shoots, and seeds; as wellas scions, rootstocks, protoplasts, calli, and the like.

As used herein, the term “polymorphism” refers to the presence of one ormore variations of a nucleic acid sequence at a locus in a population ofone or more individuals. The sequence variation can be a base or basesthat are different, inserted, or deleted. Polymorphisms can be, forexample, single nucleotide polymorphisms (SNPs), simple sequence repeats(SSRs), and Indels, which are insertions and deletions. Additionally,the variation can be in a transcriptional profile or a methylationpattern. The polymorphic sites of a nucleic acid sequence can bedetermined by comparing the nucleic acid sequences at one or more lociin two or more germplasm entries.

As used herein, the term “population” refers to a geneticallyheterogeneous collection of plants sharing a common genetic derivation.

As used herein, the term “primer” refers to an oligonucleotide which iscapable of annealing to a nucleic acid target allowing a DNA polymeraseto attach, thereby serving as a point of initiation of DNA synthesiswhen placed under conditions in which synthesis of a primer extensionproduct is induced (e.g., in the presence of nucleotides and an agentfor polymerization such as DNA polymerase and at a suitable temperatureand pH). In some embodiments, a plurality of primers are employed toamplify Zea mays nucleic acids (e.g., using the polymerase chainreaction; PCR).

As used herein, the term “probe” refers to a nucleic acid (e.g., asingle stranded nucleic acid or a strand of a double stranded or higherorder nucleic acid, or a subsequence thereof) that can form ahydrogen-bonded duplex with a complementary sequence in a target nucleicacid sequence. Typically, a probe is of sufficient length to form astable and sequence-specific duplex molecule with its complement, and assuch can be employed in some embodiments to detect a sequence ofinterest present in a plurality of nucleic acids.

As used herein, the term “progeny” refers to any plant that results froma natural or assisted breeding of one or more plants. For example,progeny plants can be generated by crossing two plants (including, butnot limited to crossing two unrelated plants, backcrossing a plant to aparental plant, intercrossing two plants, etc.), but can also begenerated by selfing a plant, creating a double haploid, or othertechniques that would be known to one of ordinary skill in the art.

As used herein, the phrase “quantitative trait locus” (QTL; quantitativetrait loci—QTLs) refers to a genetic locus (or loci) that control tosome degree a numerically representable trait that, in some embodiments,is continuously distributed. As such, the term MTL “marker trait loci”is used herein to refer to a chromosomal region containing alleles(e.g., in the form of genes or regulatory sequences) associated with theexpression of a phenotypic trait. Thus, an MTL “associated with” a yieldtrait (e.g., a starch, protein, oil, and/or ethanol production trait)refers to one or more regions located on one or more chromosomes thatincludes at least one gene the expression of which influences a level ofproduction and/or at least one regulatory region that controls theexpression of one or more genes involved in one or more yield traits.The MTLs can be defined by indicating their genetic location in thegenome of a given Zea mays plant using one or more molecular genomicmarkers. One or more markers, in turn, indicate a specific locus.Distances between loci are usually measured by the frequency ofcrossovers between loci on the same chromosome. The farther apart twoloci are, the more likely that a crossover will occur between them.Conversely, if two loci are close together, a crossover is less likelyto occur between them. Typically, one centiMorgan (cM) is equal to 1%recombination between loci. When a QTL can be indicated by multiplemarkers, the genetic distance between the end-point markers isindicative of the size of the QTL.

As used herein, the phrase “recombination” refers to an exchange of DNAfragments between two DNA molecules or chromatids of paired chromosomes(a “crossover”) over in a region of similar or identical nucleotidesequences. A “recombination event” is herein understood to refer to ameiotic crossover.

As used herein, the term “regenerate”, and grammatical variants thereof,refers to the production of a plant from tissue culture.

As used herein, the phrases “selected allele”, “desired allele”, and“allele of interest” are used interchangeably to refer to a nucleic acidsequence that includes a polymorphic allele associated with a desiredtrait. It is noted that a “selected allele”, “desired allele”, and/or“allele of interest” can be associated with either an increase in adesired trait or a decrease in a desired trait, depending on the natureof the phenotype sought to be generated in an introgressed plant.

As used herein, the phrase “single nucleotide polymorphism”, or “SNP”,refers to a polymorphism that constitutes a single base pair differencebetween two nucleotide sequences. As used herein, the term “SNP” alsorefers to differences between two nucleotide sequences that result fromsimple alterations of one sequence in view of the other that occurs at asingle site in the sequence. For example, the term “SNP” is intended torefer not just to sequences that differ in a single nucleotide as aresult of a nucleic acid substitution in one versus the other, but isalso intended to refer to sequences that differ in 1, 2, 3, or morenucleotides as a result of a deletion of 1, 2, 3, or more nucleotides ata single site in one of the sequences versus the other. It would beunderstood that in the case of two sequences that differ from each otheronly by virtue of a deletion of 1, 2, 3, or more nucleotides at a singlesite in one of the sequences versus the other, this same scenario can beconsidered an addition of 1, 2, 3, or more nucleotides at a single sitein one of the sequences versus the other, depending on which of the twosequences is considered the reference sequence. Single site insertionsand/or deletions are thus also considered to be encompassed by the term“SNP”.

As used herein, the phrase “stringent hybridization conditions” refersto conditions under which a polynucleotide hybridizes to its targetsubsequence, typically in a complex mixture of nucleic acids, but toessentially no other sequences. Stringent conditions aresequence-dependent and can be different under different circumstances.

Longer sequences typically hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, 1993. Generally, stringent conditions are selectedto be about 5-10° C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength pH. The Tm is thetemperature (under defined ionic strength, pH, and nucleic acidconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium). Exemplary stringent conditions are those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides).

Stringent conditions can also be achieved with the addition ofdestabilizing agents such as formamide. Additional exemplary stringenthybridization conditions include 50% formamide, 5×SSC, and 1% SDSincubating at 42° C.; or SSC, 1% SDS, incubating at 65° C.; with one ormore washes in 0.2×SSC and 0.1% SDS at 65° C. For PCR, a temperature ofabout 36° C. is typical for low stringency amplification, althoughannealing temperatures can vary between about 32° C. and 48° C. (orhigher) depending on primer length. Additional guidelines fordetermining hybridization parameters are provided in numerous references(see e.g., Ausubel et al., 1999).

As used herein, the phrase “TAQMAN® Assay” refers to real-time sequencedetection using PCR based on the TAQMAN® Assay sold by AppliedBiosystems, Inc. of Foster City, Calif., United States of America. Foran identified marker a TAQMAN® Assay can be developed for theapplication in the breeding program.

As used herein, the term “tester” refers to a line used in a testcrosswith one or more other lines wherein the tester and the line(s(testedare genetically dissimilar. A tester can be an isogenic line to thecrossed line.

As used herein, the term “trait” refers to a phenotype of interest, agene that contributes to a phenotype of interest, as well as a nucleicacid sequence associated with a gene that contributes to a phenotype ofinterest. For example, a “yield trait” refers to a yield phenotype aswell as a gene that contributes to a yield phenotype and a nucleic acidsequence (e.g., an SNP or other marker) that is associated with a yieldphenotype.

As used herein, the term “transgene” refers to a nucleic acid moleculeintroduced into an organism or its ancestors by some form of artificialtransfer technique. The artificial transfer technique thus creates a“transgenic organism” or a “transgenic cell”. It is understood that theartificial transfer technique can occur in an ancestor organism (or acell therein and/or that can develop into the ancestor organism) and yetany progeny individual that has the artificially transferred nucleicacid molecule or a fragment thereof is still considered transgenic evenif one or more natural and/or assisted breedings result in theartificially transferred nucleic acid molecule being present in theprogeny individual.

As used herein, the term “yield” refers to any measure of a plant, itsparts, or its structure that can be measured and/or quantitated in orderto assess an extent of or a rate of plant growth and development. Assuch, a “yield trait” is any trait that can be shown to influence yieldin a plant under any set of growth conditions. Exemplary yield traitsinclude, but are not limited to protein content, starch content, oilcontent, and digestibility (ethanol production) at 24, 48, or 72 hours,as those traits are described herein.

For example, starch content can be measured in a plant, and as set forthherein, can correlate with increased yield in Zea mays. Therefore,“starch” is a “yield trait” as that term is employed herein. Similarly,a genetic locus that is associated with increased or decreased starchcontent in a Zea mays plant (or a part thereof) is referred to herein as“associated with starch” or a “starch-associated trait”, but it is alsoa locus that is “associated with yield” and is a “yield-associatedtrait”. The same variations with respect to protein content, oilcontent, and digestibility (ethanol production) at 24, 48, or 72 hoursalso apply to the general terms and phrases “yield”, “associated withyield”, and “yield-associated” as those terms and phrases are usedherein.

II. Molecular Markers, MTLs, and Compositions for Assaying Nucleic AcidSequences

Molecular markers are used for the visualization of differences innucleic acid sequences. This visualization can be due to DNA-DNAhybridization techniques after digestion with a restriction enzyme(e.g., an RFLP) and/or due to techniques using the polymerase chainreaction (e.g., STS, SSR/microsatellites, AFLP, and the like.). In someembodiments, all differences between two parental genotypes segregate ina mapping population based on the cross of these parental genotypes. Thesegregation of the different markers can be compared and recombinationfrequencies can be calculated. Methods for mapping markers in plants aredisclosed in, for example, Glick & Thompson, 1993; Zietkiewicz et al.,1994. The recombination frequencies of molecular markers on differentchromosomes are generally 50%. Between molecular markers located on thesame chromosome, the recombination frequency generally depends on thedistance between the markers. A low recombination frequency typicallycorresponds to a small genetic distance between markers on a chromosome.Comparing all recombination frequencies results in the most logicalorder of the molecular markers on the chromosomes. This most logicalorder can be depicted in a linkage map (Paterson, 1996). A group ofadjacent or contiguous markers on the linkage map that is associatedwith increased yield can provide the position of an MTL associated withincreased yield.

IIA. Ethanol Production (Digestibility) Marker Trait Loci

In some embodiments, the presently disclosed subject matter providesmarkers associated with improved ethanol production traits, alsoreferred to herein as “digestibility” (DGST) traits at 24, 48, or 72hours. As used herein, the phrase “digestibility at 24 hours” refers todry grind ethanol percentage after 24 hours fermentation. Similarly, thephrases “digestibility at 48 hours” and “digestibility at 72 hours”refer to dry grind ethanol percentage after 48 and 72 hoursfermentation, respectively (see Table 4 below).

As set forth in Tables 5 and 6, the inbred platform and the inbred panelincluded over 1700 different lines that were tested for digestibility at24, 48, and 72 hours. Summarizing the data presented therein, the meanvalues for digestibility at 24, 48, and 72 hours were 5.02±0.92,6.34±1.08, and 7.88±1.01, respectively. As such, the presence of animproved ethanol production trait in a Zea mays plant (or in a part,progeny, or tissue culture thereof) results in the Zea mays plant havinga digestibility at 24 hours that is greater than 5.02, 6.34, and 7.88,respectively. In non-limiting, exemplary embodiments, an improveddigestibility trait can result in a plant with digestibility that is insome embodiments greater than 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0 at 24hours; in some embodiments greater than 6.5, 7.0, 7.5, 8.0, 8.5, 9.0,9.5, or 10.0 at 48 hours; and/or in some embodiments greater than 8.0,8.5, 9.0, 9.5, 10.0, 10.5, 11.0, or 11.5 at 72 hours. Alternatively, ifa reduced digestibility is desired at 24, 48, and/or 72 hours, thedigestibility of an improved plant can be in some embodiments less than5.0, 4.5, 4.0, 3.5, 3.0, 2.5, or 2.0 at 24 hours; in some embodimentsless than 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, or 1.5 at 48hours; and/or in some embodiments less than 7.5, 7.0, 6.5, 6.0, 5.5,5.0, 4.5, 4.0, 3.5, 3.0, or 2.5 at 72 hours.

In some embodiments, the markers are associated with one or more allelesthat confer an ethanol production-associated trait. In some embodiments,the one or more alleles are characterized by one or more Marker TraitLoci (MTL) selected from, but not limited to, MTL1-MTL18, which arelocated on seven (7) different chromosomes as follows:

(i) MTL1 is defined by a first assay primer and a second assay primer,wherein said assays primers amplify a subsequence of SEQ ID NO: 1generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 2 anda second assay primer comprising a nucleotide sequence as set forth inSEQ ID NO: 3; and further wherein MTL1 identifies alleles of an ethanolproduction-associated trait by identification of a single nucleicpolymorphism at nucleotide position 701 of SEQ ID NO: 1 (nucleotideposition 30 of SEQ ID NO: 111) and comprises any part of a DNA sequenceassociated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQID NO: 1 on Zea mays chromosome 5 that confers an improved ethanolproduction-associated trait;

(ii) MTL2 is defined by a first assay primer and a second assay primer,wherein said assays primers amplify a subsequence of SEQ ID NO: 4generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 5 anda second assay primer comprising a nucleotide sequence as set forth inSEQ ID NO: 6; and further wherein MTL2 identifies alleles of an ethanolproduction-associated trait by identification of a single nucleicpolymorphism at nucleotide position 498 of SEQ ID NO: 4 (nucleotideposition 23 of SEQ ID NO: 112) and comprises any part of a DNA sequenceassociated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQID NO: 1 on Zea mays chromosome 5 that confers an improved ethanolproduction-associated trait;

(iii) MTL3 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 7generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 8 anda second assay primer comprising a nucleotide sequence as set forth inSEQ ID NO: 9; and further wherein MTL3 identifies alleles of an ethanolproduction-associated trait by identification of a single nucleicpolymorphism at nucleotide position 587 of SEQ ID NO: 7 (nucleotideposition 33 of SEQ ID NO: 113) and comprises any part of a DNA sequenceassociated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQID NO: 7 on Zea mays chromosome 5 that confers an improved ethanolproduction-associated trait;

(iv) MTL4 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 10generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 11and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 12; and further wherein MTL4 identifies alleles of anethanol production-associated trait by identification of a singlenucleic polymorphism at nucleotide position 708 of SEQ ID NO: 10(nucleotide position 76 of SEQ ID NO: 114) and comprises any part of aDNA sequence associated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of25 cM of SEQ ID NO: 10 on Zea mays chromosome 3 that confers an improvedethanol production-associated trait;

(v) MTL5 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 13generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 14and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 15; and further wherein MTL5 identifies alleles of anethanol production-associated trait by identification of a singlenucleic polymorphism at nucleotide position 140 of SEQ ID NO: 13(nucleotide position 58 of SEQ ID NO: 115) and comprises any part of aDNA sequence associated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of25 cM of SEQ ID NO: 13 on Zea mays chromosome 2 that confers an improvedethanol production-associated trait;

(vi) MTL6 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 16generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 17and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 18; and further wherein MTL16 identifies alleles of anethanol production-associated trait by identification of a singlenucleic polymorphism at nucleotide position 116 of SEQ ID NO: 16(nucleotide position 33 of SEQ ID NO: 116) and comprises any part of aDNA sequence associated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of25 cM of SEQ ID NO: 16 on Zea mays chromosome 5 that confers an improvedethanol production-associated trait;

(vii) MTL7 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 19generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 20and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 21; and further wherein MTL7 identifies alleles of anethanol production-associated trait by identification of a singlenucleic polymorphism at nucleotide position 269 of SEQ ID NO: 19(nucleotide position 32 of SEQ ID NO: 117) and comprises any part of aDNA sequence associated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of25 cM of SEQ ID NO: 19 on Zea mays chromosome 7 that confers an improvedethanol production-associated trait;

(viii) MTL8 is defined by a first assay primer and a second assayprimer, wherein said assay primers amplify a subsequence of SEQ ID NO:22 generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 23and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 24; and further wherein MTL8 identifies alleles of anethanol production-associated trait by identification of a singlenucleic polymorphism at nucleotide position 280 of SEQ ID NO: 22(nucleotide position 23 of SEQ ID NO: 118) and comprises any part of aDNA sequence associated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of25 cM of SEQ ID NO: 22 on Zea mays chromosome 5 that confers an improvedethanol production-associated trait;

(ix) MTL9 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 25generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 26and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 27; and further wherein MTL9 identifies alleles of anethanol production-associated trait by identification of a singlenucleic polymorphism at nucleotide position 374 of SEQ ID NO: 25(nucleotide position 46 of SEQ ID NO: 119) and comprises any part of aDNA sequence associated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of25 cM of SEQ ID NO: 25 on Zea mays chromosome 5 that confers an improvedethanol production-associated trait;

(x) MTL10 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 28generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 29and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 30; and further wherein MTL10 identifies alleles of anethanol production-associated trait by identification of a singlenucleic polymorphism at nucleotide position 236 of SEQ ID NO: 28(nucleotide position 41 of SEQ ID NO: 120) and comprises any part of aDNA sequence associated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of25 cM of SEQ ID NO: 28 on Zea mays chromosome 1 that confers an improvedethanol production-associated trait;

(xi) MTL11 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 31generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 32and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 33; and further wherein MTL11 identifies alleles of anethanol production-associated trait by identification of a singlenucleic polymorphism at nucleotide position 605 of SEQ ID NO: 31(nucleotide position 32 of SEQ ID NO: 121) and comprises any part of aDNA sequence associated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of25 cM of SEQ ID NO: 31 on Zea mays chromosome 2 that confers an improvedethanol production-associated trait;

(xii) MTL12 is defined by a first assay primer and a second assayprimer, wherein said assay primers amplify a subsequence of SEQ ID NO:34 generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 35and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 36; and further wherein MTL12 identifies alleles of anethanol production-associated trait by identification of a singlenucleotide polymorphism at nucleotide position 349 of SEQ ID NO: 34(nucleotide position 48 of SEQ ID NO: 122) and comprises any part of aDNA sequence associated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of25 cM of SEQ ID NO: 34 on Zea mays chromosome 10 that confers animproved ethanol production-associated trait;

(xiii) MTL13 is defined by a first assay primer and a second assayprimer, wherein said assay primers amplify a subsequence of SEQ ID NO:37 generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 38and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 39; and further wherein MTL13 identifies alleles of anethanol production-associated trait by identification of a singlenucleic polymorphism at nucleotide position 389 of SEQ ID NO: 37(nucleotide position 45 of SEQ ID NO: 123) and comprises any part of aDNA sequence associated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of25 cM of SEQ ID NO: 37 on Zea mays chromosome 8 that confers an improvedethanol production-associated trait;

(xiv) MTL14 is defined by a first assay primer and a second assayprimer, wherein said assay primers amplify a subsequence of SEQ ID NO:40 generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 41and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 42; and further wherein MTL14 identifies alleles of anethanol production-associated trait by identification of a singlenucleic polymorphism at nucleotide position 66 of SEQ ID NO: 40(nucleotide position 44 of SEQ ID NO: 124) and comprises any part of aDNA sequence associated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of25 cM of SEQ ID NO: 40 on Zea mays chromosome 1 that confers an improvedethanol production-associated trait;

(xv) MTL15 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 43generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 44and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 45; and further wherein MTL15 identifies alleles of anethanol production-associated trait by identification of a singlenucleic polymorphism at nucleotide position 278 of SEQ ID NO: 43(nucleotide position 48 of SEQ ID NO: 125) and comprises any part of aDNA sequence associated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of25 cM of SEQ ID NO: 43 on Zea mays chromosome 5 that confers an improvedethanol production-associated trait;

(xvi) MTL16 is defined by a first assay primer and a second assayprimer, wherein said assay primers amplify a subsequence of SEQ ID NO:46 generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 47and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 48; and further wherein MTL16 identifies alleles of anethanol production-associated trait by identification of a singlenucleic polymorphism at nucleotide position 463 of SEQ ID NO: 46(nucleotide position 20 of SEQ ID NO: 126) and comprises any part of aDNA sequence associated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of25 cM of SEQ ID NO: 46 on Zea mays chromosome 1 that confers an improvedethanol production-associated trait;

(xvii) MTL17 is defined by a first assay primer and a second assayprimer, wherein said assay primers amplify a subsequence of SEQ ID NO:49 generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 50and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 51; and further wherein MTL17 identifies alleles of anethanol production-associated trait by identification of a singlenucleic polymorphism at nucleotide position 510 of SEQ ID NO: 49(nucleotide position 126 of SEQ ID NO: 127) and comprises any part of aDNA sequence associated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of25 cM of SEQ ID NO: 49 on Zea mays chromosome 5 that confers an improvedethanol production-associated trait; and

(xviii) MTL18 is defined by a first assay primer and a second assayprimer, wherein said assay primers amplify a subsequence of SEQ ID NO:52 generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 53and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 54; and further wherein MTL18 identifies alleles of anethanol production-associated trait by identification of a singlenucleic polymorphism at nucleotide position 134 of SEQ ID NO: 52(nucleotide position 126 of SEQ ID NO: 128) and comprises any part of aDNA sequence associated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of25 cM of SEQ ID NO: 52 on Zea mays chromosome 10 that confers animproved ethanol production-associated trait.

II.B. Protein Marker Trait Loci

In some embodiments, the presently disclosed subject matter providesmarkers associated with improved protein traits.

As used herein, the phrase “protein content” refers to kernel proteincontent measured in percentage. As set forth in Tables 5 and 6, theinbred platform and the inbred panel included over 1700 different linesthat were tested for protein content. Summarizing the data presentedtherein, the mean value for protein content was 12.45±1.46. As such, thepresence of an improved protein trait in a Zea mays plant (or in a part,progeny, or tissue culture thereof) can result in the Zea mays planthaving a protein content that is greater than 12.45. In non-limiting,exemplary embodiments, the protein content of an improved Zea mays plantcan be in some embodiments greater than 12.5, 13.0, 13.5, 14.0, 14.5,15.0, 15.5, 16.0, 16.5, 17.0, or 17.5. Alternatively, if a reducedprotein content is desired, the protein content of an improved Zea maysplant can be in some embodiments less than 12.0, 11.5, 11.0, 10.5, 10.0,9.5, 9.0, or 8.5.

In some embodiments, the markers are associated with one or more allelesthat confer a protein associated trait. In some embodiments, the one ormore alleles are characterized by one or more Marker Trait (Protein)Loci (MTPL) selected from, but not limited to, MTPL1-MTPL10, which arelocated on seven (7) different chromosomes as follows:

(i) MTPL1 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 1generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 2 anda second assay primer comprising a nucleotide sequence as set forth inSEQ ID NO: 3; and further wherein MTPL1 identifies alleles of a proteinassociated trait by identification of a single nucleic polymorphism atnucleotide position 701 of SEQ ID NO: 1 (nucleotide position 30 of SEQID NO: 111) and comprises any part of a DNA sequence associated within1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQ ID NO: 1 on Zeamays chromosome 5 that confers a protein associated trait;

(ii) MTPL2 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 10generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 11and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 12; and further wherein MTPL2 identifies alleles of aprotein associated trait by identification of a single nucleicpolymorphism at nucleotide position 708 of SEQ ID NO: 10 (nucleotideposition 76 of SEQ ID NO: 114) and comprises any part of a DNA sequenceassociated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQID NO: 10 on Zea mays chromosome 3 that confers a protein associatedtrait;

(iii) MTPL3 is defined by a first assay primer and a second assayprimer, wherein said assay primers amplify a subsequence of SEQ ID NO:19 generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 20and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 21; and further wherein MTPL3 identifies alleles of aprotein associated trait by identification of a single nucleicpolymorphism at nucleotide position 269 of SEQ ID NO: 19 (nucleotideposition 32 of SEQ ID NO: 117) and comprises any part of a DNA sequenceassociated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQID NO: 19 on Zea mays chromosome 7 that confers a protein associatedtrait;

(iv) MTPL4 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 22generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 23and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 24; and further wherein MTPL4 identifies alleles of aprotein associated trait by identification of a single nucleicpolymorphism at nucleotide position 280 of SEQ ID NO: 22 (nucleotideposition 23 of SEQ ID NO: 118) and comprises any part of a DNA sequenceassociated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQID NO: 22 on Zea mays chromosome 5 that confers a protein associatedtrait;

(v) MTPL5 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 58generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 59and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 60; and further wherein MTPL5 identifies alleles of aprotein associated trait by identification of a single nucleicpolymorphism at nucleotide position 119 of SEQ ID NO: 58 (nucleotideposition 23 of SEQ ID NO: 130) and comprises any part of a DNA sequenceassociated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQID NO: 58 on Zea mays chromosome 4 that confers a protein associatedtrait;

(vi) MTPL6 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 64generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 65and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 66; and further wherein MTPL6 identifies alleles of aprotein associated trait by identification of a single nucleicpolymorphism at nucleotide position 356 of SEQ ID NO: 64 (nucleotideposition 43 of SEQ ID NO: 132) and comprises any part of a DNA sequenceassociated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQID NO: 64 on Zea mays chromosome 7 that confers a protein associatedtrait;

(vii) MTPL7 is defined by a first assay primer and a second assayprimer, wherein said assay primers amplify a subsequence of SEQ ID NO:52 generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 53and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 54; and further wherein MTPL7 identifies alleles of aprotein associated trait by identification of a single nucleicpolymorphism at nucleotide position 134 of SEQ ID NO: 52 (nucleotideposition 126 of SEQ ID NO: 128) and comprises any part of a DNA sequenceassociated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQID NO: 52 on Zea mays chromosome 10 that confers a protein associatedtrait;

(viii) MTPL8 is defined by a first assay primer and a second assayprimer, wherein said assay primers amplify a subsequence of SEQ ID NO:49 generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 50and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 51; and further wherein MTPL8 identifies alleles of aprotein associated trait by identification of a single nucleicpolymorphism at nucleotide position 510 of SEQ ID NO: 49 (nucleotideposition 126 of SEQ ID NO: 127) and comprises any part of a DNA sequenceassociated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQID NO: 49 on Zea mays chromosome 5 that confers a protein associatedtrait;

(ix) MTPL9 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 40generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 41and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 42; and further wherein MTPL9 identifies alleles of aprotein associated trait by identification of a single nucleicpolymorphism at nucleotide position 66 of SEQ ID NO: 40 (nucleotideposition 44 of SEQ ID NO: 123) and comprises any part of a DNA sequenceassociated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQID NO: 40 on Zea mays chromosome 1 that confers a protein associatedtrait; and

(x) MTPL10 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 61generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 62and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 63; and further wherein MTSPL10 identifies alleles of aprotein associated trait by identification of a single nucleicpolymorphism at nucleotide position 347 of SEQ ID NO: 61 (nucleotideposition 53 of SEQ ID NO: 131) and comprises any part of a DNA sequenceassociated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQID NO: 61 on Zea mays chromosome 6 that confers a protein associatedtrait.

II.C. Starch Marker Trait Loci

In some embodiments, the presently disclosed subject matter providesmarkers associated with improved starch traits.

As used herein, the phrase “starch content” refers to grain starchcontent measured in percentage. As set forth in Tables 5 and 6, theinbred platform and the inbred panel included over 1700 different linesthat were tested for starch content. Summarizing the data presentedtherein, the mean value for starch content was 70.03±2.37. As such, thepresence of an improved starch trait in a Zea mays plant (or in a part,progeny, or tissue culture thereof) can result in the Zea mays planthaving a starch content that is greater than 70.03. In non-limiting,exemplary embodiments, the starch content of an improved Zea mays plantcan be in some embodiments greater than 71, 72, 73, 74, 75, 76, 77, or78. Alternatively, if a reduced starch content is desired, the starchcontent of an improved Zea mays plant can be in some embodiments lessthan 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, or 60.

In some embodiments, the markers are associated with one or more allelesthat confer a starch associated trait. In some embodiments, the one ormore alleles are characterized by one or more Marker Trait (Starch) Loci(MTSL) selected from, but not limited to, MTSL1-MTSL6, which are locatedon five (5) different chromosomes as follows:

(i) MTSL1 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 1generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 2 anda second assay primer comprising a nucleotide sequence as set forth inSEQ ID NO: 3; and further wherein MTSL1 identifies alleles of a starchassociated trait by identification of a single nucleic polymorphism atnucleotide position 701 of SEQ ID NO: 1 (nucleotide position 30 of SEQID NO: 111) and comprises any part of a DNA sequence associated within1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQ ID NO: 1 on Zeamays chromosome 5 that confers a starch associated trait;

(ii) MTSL2 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 4generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 5 anda second assay primer comprising a nucleotide sequence as set forth inSEQ ID NO: 6; and further wherein MTSL2 identifies alleles of a starchassociated trait by identification of a single nucleic polymorphism atnucleotide position 498 of SEQ ID NO: 4 (nucleotide position 23 of SEQID NO: 112) and comprises any part of a DNA sequence associated within1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQ ID NO: 4 on Zeamays chromosome 5 that confers a starch associated trait;

(iii) MTSL3 is defined by a first assay primer and a second assayprimer, wherein said assay primers amplify a subsequence of SEQ ID NO: 7generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 8 anda second assay primer comprising a nucleotide sequence as set forth inSEQ ID NO: 9; and further wherein MTSL3 identifies alleles of a starchassociated trait by identification of a single nucleic polymorphism atnucleotide position 587 of SEQ ID NO: 7 (nucleotide position 33 of SEQID NO: 113) and comprises any part of a DNA sequence associated within1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQ ID NO: 7 on Zeamays chromosome 5 that confers a starch associated trait;

(iv) MTSL4 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 10generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 11and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 12; and further wherein MTSL4 identifies alleles of astarch associated trait by identification of a single nucleicpolymorphism at nucleotide position 708 of SEQ ID NO: 10 (nucleotideposition 76 of SEQ ID NO: 114) and comprises any part of a DNA sequenceassociated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQID NO: 10 on Zea mays chromosome 3 that confers a starch associatedtrait;

(v) MTSL5 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 37generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 38and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 39; and further wherein MTSL5 identifies alleles of astarch associated trait by identification of a single nucleicpolymorphism at nucleotide position 389 of SEQ ID NO: 37 (nucleotideposition 45 of SEQ ID NO: 123) and comprises any part of a DNA sequenceassociated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQID NO: 37 on Zea mays chromosome 8 that confers a starch associatedtrait; and

(vi) MTSL6 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 61generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 62and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 63; and further wherein MTSL6 identifies alleles of astarch associated trait by identification of a single nucleicpolymorphism at nucleotide position 347 of SEQ ID NO: 61 (nucleotideposition 53 of SEQ ID NO: 131) and comprises any part of a DNA sequenceassociated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQID NO: 61 on Zea mays chromosome 6 that confers a starch associatedtrait.

II.D. Oil Marker Trait Loci

In some embodiments, the presently disclosed subject matter providesmarkers associated with improved oil traits. In some embodiments, themarkers are associated with one or more alleles that confer an oilassociated trait.

As used herein, the phrase “oil content” refers to grain oil contentmeasured in percentage. As set forth in Tables 5 and 6, the inbredplatform and the inbred panel included over 1700 different lines thatwere tested for oil content. Summarizing the data presented therein, themean value for oil content was 3.93±0.62. As such, the presence of animproved oil trait in a Zea mays plant (or in a part, progeny, or tissueculture thereof) can result in the Zea mays plant having an oil contentthat is greater than 3.93. In non-limiting, exemplary embodiments, theoil content of an improved Zea mays plant can be in some embodimentsgreater than 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5. Alternatively, if areduced oil content is desired, the oil content of an improved Zea maysplant can be in some embodiments less than 3.5, 3.0, or 2.5.

In some embodiments, the one or more alleles are characterized by one ormore Marker Trait (Oil) Loci (MTOL) selected from, but not limited to,MTOL1-MTOL3, which are located on two (2) different chromosomes asfollows:

(i) MTOL1 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 55generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 56and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 57; and further wherein MTOL1 identifies alleles of an oilassociated trait by identification of a single nucleic polymorphism atnucleotide position 367 of SEQ ID NO: 55 (nucleotide position 32 of SEQID NO: 129) and comprises any part of a DNA sequence associated within1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQ ID NO: 55 on Zeamays chromosome 1 that confers an oil associated trait;

(ii) MTOL2 is defined by a first assay primer and a second assay primer,wherein said assay primers amplify a subsequence of SEQ ID NO: 64generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 65and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 66; and further wherein MTOL2 identifies alleles of an oilassociated trait by identification of a single nucleic polymorphism atnucleotide position 356 of SEQ ID NO: 64 (nucleotide position 43 of SEQID NO: 132) and comprises any part of a DNA sequence associated within1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, of 25 cM of SEQ ID NO: 64 on Zeamays chromosome 7 that confers an oil associated trait; and

(iii) MTOL3 is defined by a first assay primer and a second assayprimer, wherein said assay primers amplify a subsequence of SEQ ID NO:28 generated by amplifying a Zea mays nucleic acid with a first assayprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 29and a second assay primer comprising a nucleotide sequence as set forthin SEQ ID NO: 30; and further wherein MTOL3 identifies alleles of an oilassociated trait by identification of a single nucleic polymorphism atnucleotide position 236 of SEQ ID NO: 28 (nucleotide position 41 of SEQID NO: 120) and comprises any part of a DNA sequence associated within1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 cM of SEQ ID NO: 28 on Zeamays chromosome 1 that confers an oil associated trait.

In some embodiments, a DNA sequence associated within 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, or 25 cM of a marker of the presently disclosedsubject matter displays a genetic recombination frequency of less thanabout 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% with themarker of the presently disclosed subject matter. In some embodiments,the one or more marker loci associated with improved yield (e.g.,starch, protein, oil, and/or ethanol production) traits are selectedfrom the marker loci of increased starch and lower levels of protein. Insome embodiments, the one or more marker loci associated with improvedyield traits are a plurality of loci selected from the marker loci ofstarch and ethanol production traits, protein and ethanol productiontraits, or any other combination of two or more of the yield traitsdisclosed herein. In some embodiments, the one or more marker lociassociated with improved yield traits are selected from marker locilocalizing within the chromosome intervals of 25 cM or less. In someembodiments, the germplasm is a Zea mays line or variety.

The presently disclosed subject matter thus provides in some embodimentsisolated and purified genetic markers associated with improved yield(e.g., starch, protein, oil, and/or ethanol production) traits in Zeamays. In some embodiments, the markers (a) are associated with starch,protein, oil, and/or digestibility (ethanol production) traits; or (b)comprise a nucleotide sequence that comprises the full length sequenceof any of SEQ ID NOs: 1-173, the complement of any of SEQ ID NOs: 1-173,or informative fragments thereof; or (c) comprise a nucleotide sequenceof at least 10, 15, 20, 25, or more contiguous nucleotides up to thefull length of an amplification product from a DNA sample isolated froma maize, wherein the amplification product is produced by anamplification reaction using pairs of oligonucleotide primers comprisingthe following nucleotide sequences: SEQ ID NOs: 2 and 3; SEQ ID NOs: 5and 6; SEQ ID SEQ ID NOs: 8 and 9; SEQ ID NOs: 11 and 12; SEQ ID NOs: 14and 15; SEQ ID NOs: 17 and 18; SEQ ID NOs: 20 and 21; SEQ ID NOs: 23 and24; SEQ ID NOs: 26 and 27; or SEQ ID NOs: 29 and 30; SEQ ID NOs: 32 and33; SEQ ID NOs: 35 and 36; SEQ ID NOs: 38 and 39; SEQ ID NOs: 41 and 42;SEQ ID NOs: 44 and 45; SEQ ID NOs: 47 and 48; SEQ ID NOs: 50 and 51; SEQID NOs: 53 and 54; SEQ ID NOs: 56 and 57; SEQ ID NOs: 59 and 60; SEQ IDNOs: 62 and 63; or SEQ ID NOs: 65 and 66. In some embodiments, the probecomprises an isolated and purified genetic marker as disclosed hereinand a detectable moiety.

The markers identified herein can be used is various aspects of thepresently disclosed subject matter as set forth herein. Aspects of thepresently disclosed subject matter are not to be limited to the use ofthe markers identified herein, however. It is stressed that the aspectscan also make use of markers not explicitly disclosed herein or even yetto be identified. Other than the genetic unit “gene”, on which thephenotypic expression depends on a large number of factors that cannotbe predicted, the genetic unit “MTL” denotes a region on the genome thatis directly related to a phenotypic quantifiable trait.

DNA fragments associated with the presence of an MTL including, but notlimited to MTL1-18, MTPL1-10, MTSL1-6, and MTOL1-3, are also provided.In some embodiments, the DNA fragments associated with the presence ofan MTL have a predicted length and/or nucleic acid sequence, anddetecting a DNA fragment having the predicted length and/or thepredicted nucleic acid sequence is performed such that the amplified DNAfragment has a length that corresponds (plus or minus a few bases; e.g.,a length of one, two or three bases more or less) to the predictedlength. In some embodiments, a DNA fragment is an amplified fragment andthe amplified fragment has a predicted length and/or nucleic acidsequence as does an amplified fragment produced by a similar reactionwith the same primers with the DNA from the plant in which the markerwas first detected or the nucleic acid sequence that corresponds (i.e.,as a nucleotide sequence identity of more than 80%, 90%, 95%, 97%, or99%) to the expected sequence as based on the sequence of the markerassociated with that MTL in the plant in which the marker was firstdetected. Upon a review of the instant disclosure, one of ordinary skillin the art would appreciate that markers that are absent in plants whilethey were present in at least one parent plant (so-calledtrans-markers), can also be useful in assays for detecting a desiredtrait in an progeny plant, although testing for the absence of a markerto detect the presence of a specific trait is not optimal. The detectingof an amplified DNA fragment having the predicted length or thepredicted nucleic acid sequence can be performed by any of a number oftechniques, including but not limited to standard gel electrophoresistechniques and/or by using automated DNA sequencers. The methods are notdescribed here in detail as they are well known to the skilled person.

The primer (in some embodiments an extension primer and in someembodiments an amplification primer) is in some embodiments singlestranded for maximum efficiency in extension and/or amplification. Insome embodiments, the primer is an oligodeoxyribonucleotide. A primer istypically sufficiently long to prime the synthesis of extension and/oramplification products in the presence of the agent for polymerization.The minimum lengths of the primers can depend on many factors, includingbut not limited to temperature and composition (A/T vs. G/C content) ofthe primer.

In the context of an amplification primer, these are typically providedas one or more sets of bidirectional primers that include one or moreforward and one or more reverse primers as commonly used in the art ofDNA amplification such as in PCR amplification, As such, it will beunderstood that the term “primer”, as used herein, can refer to morethan one primer, particularly in the case where there is some ambiguityin the information regarding the terminal sequence(s) of the targetregion to be amplified. Hence, a “primer” can include a collection ofprimer oligonucleotides containing sequences representing the possiblevariations in the sequence or includes nucleotides which allow a typicalbase pairing. Primers can be prepared by any suitable method. Methodsfor preparing oligonucleotides of specific sequence are known in theart, and include, for example, cloning, and restriction of appropriatesequences and direct chemical synthesis. Chemical synthesis methods caninclude, for example, the phospho di- or tri-ester method, thediethylphosphoramidate method and the solid support method disclosed inU.S. Pat. No. 4,458,068.

Primers can be labeled, if desired, by incorporating detectable moietiesby for instance spectroscopic, fluorescence, photochemical, biochemical,immunochemical, or chemical moieties.

Template-dependent extension of an oligonucleotide primer is catalyzedby a polymerizing agent in the presence of adequate amounts of the fourdeoxyribonucleotides triphosphates (dATP, dGTP, dCTP and dTTP; i.e.,dNTPs) or analogues, in a reaction medium that comprises appropriatesalts, metal cations, and a pH buffering system. Suitable polymerizingagents are enzymes known to catalyze primer- and template-dependent DNAsynthesis. Known DNA polymerases include, for example, E. coli DNApolymerase or its Klenow fragment, T4 DNA polymerase, and Taq DNApolymerase, as well as various modified versions thereof. The reactionconditions for catalyzing DNA synthesis with these DNA polymerases areknown in the art. The products of the synthesis are duplex moleculesconsisting of the template strands and the primer extension strands,which include the target sequence. These products, in turn, can serve astemplate for another round of replication. In the second round ofreplication, the primer extension strand of the first cycle is annealedwith its complementary primer; synthesis yields a “short” product whichis bound on both the 5′- and the 3′-ends by primer sequences or theircomplements. Repeated cycles of denaturation, primer annealing, andextension can result in the exponential accumulation of the targetregion defined by the primers. Sufficient cycles are run to achieve thedesired amount of polynucleotide containing the target region of nucleicacid. The desired amount can vary, and is determined by the functionwhich the product polynucleotide is to serve.

The PCR method is well described in handbooks and known to the skilledperson. After amplification by PCR, the target polynucleotides can bedetected by hybridization with a probe polynucleotide which forms astable hybrid with that of the target sequence under stringent tomoderately stringent hybridization and wash conditions. If it isexpected that the probes will be essentially completely complementary(i.e., about 99% or greater) to the target sequence, stringentconditions can be used. If some mismatching is expected, for example ifvariant strains are expected with the result that the probe will not becompletely complementary, the stringency of hybridization can bereduced. In some embodiments, conditions are chosen to rule outnon-specific/adventitious binding. Conditions that affect hybridization,and that select against non-specific binding are known in the art, andare described in, for example, Sambrook & Russell, 2001. Generally,lower salt concentration and higher temperature increase the stringencyof hybridization conditions.

In order to detect in a plant the presence of two MTLs on a singlechromosome, chromosome painting methods can also be used. In suchmethods at least a first MTL and at least a second MTL can be detectedin the same chromosome by in situ hybridization or in situ PCRtechniques. More conveniently, the fact that two MTLs are present on asingle chromosome can be confirmed by determining that they are incoupling phase: i.e., that the traits show reduced segregation whencompared to genes residing on separate chromosomes.

The groups (e.g., starch, protein, oil, and ethanol production) of MTLsidentified herein are located on a number of different chromosomes orlinkage groups and their locations can be characterized by a number ofotherwise arbitrary markers. In the present investigations, singlenucleotide polymorphisms (SNPs), were used, although restrictionfragment length polymorphism (RFLP) markers, amplified fragment lengthpolymorphism (AFLP) markers, microsatellite markers (e.g., SSRs),insertion mutation markers, sequence-characterized amplified region(SCAR) markers, cleaved amplified polymorphic sequence (CAPS) markers,isozyme markers, microarray-based technologies, TAQMAN® Assays,ILLUMINA® GOLDENGATE® Assay analysis, nucleic acid sequencingtechnologies, or combinations of these markers might also have beenused, and indeed can be used.

In general, providing complete sequence information for an MTL isunnecessary, as the way in which the MTL is first detected—through anobserved correlation between the presence of a single nucleotidepolymorphism and the presence of a particular phenotypic trait—allowsone to trace among a population of progeny plants those plants that havethe genetic potential for exhibiting a particular phenotypic trait. Byproviding a non-limiting list of markers, the presently disclosedsubject matter thus provides for the effective use of the presentlydisclosed MTLs in a breeding program. In some embodiments, a marker isspecific for a particular line of descent. Thus, a specific trait can beassociated with a particular marker.

The markers as disclosed herein not only indicate the location of theMTL, they also correlate with the presence of the specific phenotypictrait in a plant. It is noted that a single nucleotide polymorphism thatindicates where an MTL is present in the genome is non-limiting. Ingeneral, the location of an MTL is indicated by a single nucleotidepolymorphism that exhibit statistical correlation to the phenotypictrait. Once a marker is found outside a single nucleotide polymorphism(i.e., one that has a LOD-score below a certain threshold, indicatingthat the marker is so remote that recombination in the region betweenthat marker and the MTL occurs so frequently that the presence of themarker does not correlate in a statistically significant manner to thepresence of the phenotype), the boundaries of the MTL can be consideredset. Thus, it is also possible to indicate the location of the MTL byother markers located within that specified region. It is further notedthat a single nucleotide polymorphism can also be used to indicate thepresence of the MTL (and thus of the phenotype) in an individual plant,which in some embodiments means that it can be used in marker-assistedselection (MAS) procedures.

In principle, the number of potentially useful markers can be verylarge. Any marker that is linked to an MTL (e.g., falling within thephysically boundaries of the genomic region spanned by the markershaving established LOD scores above a certain threshold therebyindicating that no or very little recombination between the marker andthe MTL occurs in crosses, as well as any marker in linkagedisequilibrium to the MTL, as well as markers that represent the actualcausal mutations within the MTL) can be used in the presently disclosedmethods and compositions, and are within the scope of the presentlydisclosed subject matter. This means that the markers identified in theapplication as associated with the MTLs (e.g., MTL1-MTL18, MTPL1-10,MTSL1-6, and MTOL1-3) are non-limiting examples of markers suitable foruse in the presently disclosed methods and compositions. Moreover, whenan MTL, or the specific trait-conferring part thereof, is introgressedinto another genetic background (i.e., into the genome of another maizeor another plant species), then some markers might no longer be found inthe progeny although the trait is present therein, indicating that suchmarkers are outside the genomic region that represents the specifictrait-conferring part of the MTL in the original parent line only andthat the new genetic background has a different genomic organization.Such markers of which the absence indicates the successful introductionof the genetic element in the progeny are called “trans markers” and canbe equally suitable with respect to the presently disclosed subjectmatter.

Upon the identification of an MTL, the MTL effect (e.g., the trait) canfor instance be confirmed by assessing trait in progeny segregating forthe MTLs under investigation. The assessment of the trait can suitablybe performed by using phenotypic assessment as known in the art foryield traits. For example, NIR can be employed to detect oil, ethanol,starch, and protein. Additionally, (field) trials under natural and/orirrigated conditions can be conducted to assess the traits of hybridand/or inbred maize.

The markers provided by the presently disclosed subject matter can beused for detecting the presence of one or more yield trait alleles atMTLs of the presently disclosed subject matter in a suspected yieldtrait introgressed maize plant, and can therefore be used in methodsinvolving marker-assisted breeding and selection of such yield traitbearing maize plants. In some embodiments, detecting the presence of anMTL of the presently disclosed subject matter is performed with at leastone of the markers for an MTL as defined herein. The presently disclosedsubject matter therefore relates in another aspect to a method fordetecting the presence of an MTL for at least one of the presentlydisclosed yield traits, comprising detecting the presence of a nucleicacid sequence of the MTL in a trait bearing maize plant, which presencecan be detected by the use of the disclosed markers.

In some embodiments, the detecting comprises determining the nucleotidesequence of a Zea mays nucleic acid associated with an MTL. Thenucleotide sequence of an MTL of the presently disclosed subject mattercan for instance be resolved by determining the nucleotide sequence ofone or more markers associated with the MTL and designing internalprimers for the marker sequences that can then be used to furtherdetermine the sequence of the MTL outside of the marker sequences. Forinstance, the nucleotide sequence of the SNP markers disclosed hereincan be obtained by isolating the markers from the electrophoresis gelused in the determination of the presence of the markers in the genomeof a subject plant, and determining the nucleotide sequence of themarkers by, for example, dideoxy chain termination sequencing methods,which are well known in the art. In some embodiments of such methods fordetecting the presence of an MTL in a trait bearing maize plant, themethod can also comprise providing a oligonucleotide or polynucleotidecapable of hybridizing under stringent hybridization conditions to anucleic acid sequence of a marker linked to the MTL, in some embodimentsselected from the markers disclosed herein, contacting theoligonucleotide or polynucleotide with digested genomic nucleic acid ofa trait bearing maize plant, and determining the presence of specifichybridization of the oligonucleotide or polynucleotide to the digestedgenomic nucleic acid. In some embodiments, the method is performed on anucleic acid sample obtained from the trait-bearing maize plant,although in situ hybridization methods can also be employed.Alternatively, one of ordinary skill in the art can, once the nucleotidesequence of the MTL has been determined, design specific hybridizationprobes or oligonucleotides capable of hybridizing under stringenthybridization conditions to the nucleic acid sequence of the MTL and canuse such hybridization probes in methods for detecting the presence ofan MTL disclosed herein in a trait bearing maize plant.

The presently disclosed subject matter also provides compositionscomprising amplification primer pairs capable of initiatingpolymerization by a nucleic acid polymerase on Zea mays nucleic acidtemplates to generate Zea mays marker amplicons. In some embodiments,the Zea mays marker amplicons correspond to SEQ ID NOs: 1, 4, 7, 10, 13,16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, and 64,and/or informative fragments thereof. As used herein, the phrase“informative fragment” refers to a nucleotide sequence of any lengththat is present within any of SEQ ID NOs: 1-173 (e.g., at least 10, 15,20, 25, or more nucleotides up to and including the full length of anyof SEQ ID NOs: 1-173) that is indicative of the presence or absence of agenetic marker associated with improved yield (e.g., starch, protein,oil, and/or ethanol production) traits in Zea mays. In some embodiments,an informative fragment comprises an SNP selected from among nucleotideposition 701 of SEQ ID NO: 1; nucleotide position 498 of SEQ ID NO: 4;nucleotide position 587 of SEQ ID NO: 7; nucleotide position 708 of SEQID NO: 10; nucleotide position 140 of SEQ ID NO: 13; nucleotide position116 of SEQ ID NO: 16; nucleotide position 269 of SEQ ID NO: 19;nucleotide position 280 of SEQ ID NO: 22; nucleotide position 374 of SEQID NO: 25; nucleotide position 236 of SEQ ID NO: 28; nucleotide position605 of SEQ ID NO: 31; nucleotide position 349 of SEQ ID NO: 34;nucleotide position 389 of SEQ ID NO: 37; nucleotide position 66 of SEQID NO: 40; nucleotide position 278 of SEQ ID NO: 43; nucleotide position463 of SEQ ID NO: 46; nucleotide position 510 of SEQ ID NO: 49;nucleotide position 134 of SEQ ID NO: 52; nucleotide position 367 of SEQID NO: 55; nucleotide position 119 of SEQ ID NO: 58; nucleotide position347 of SEQ ID NO: 61; and nucleotide position 356 of SEQ ID NO: 64.

III. Methods for Employing Markers and MTLs to Produce Improved MaizePlants by Marker Assisted Selection and Marker Assisted Breeding

The presently disclosed subject matter provides methods for conveyingselected yield traits (e.g., selected starch, protein, oil, and/orethanol production traits) into maize germplasm. In some embodiments,the methods comprise introgressing yield traits into maize using one ormore nucleic acid markers for marker-assisted selection among maizelines to be used in a maize breeding program, wherein the markers arelinked to yield traits. In some embodiments, the one or more nucleicacid markers are selected from the group including, but not limited to,markers for starch, protein, oil, and/or ethanol production traits. Insome embodiments, the one or more nucleic acid markers are selected fromthe group of markers listed in SEQ ID NOs: 1-173. In some embodiments,the marker-assisted selection comprises the use of an analysis techniqueselected from the group including, but not limited to, single nucleotidepolymorphism (SNP) analysis, random amplified polymorphic DNA (RAPD)analysis, restriction fragment-length polymorphism (RFLP) analysis,microsatellite analysis, amplified fragment length polymorphism (AFLP)analysis, TAQMAN® Assay analysis (Applied Biosystems, Inc., Foster City,Calif., United States of America), and ILLUMINA® GOLDENGATE® GenotypingAssay analysis (Illumina Inc., San Diego, Calif., United States ofAmerica). In some embodiments, the methods further comprise screening anintrogressed maize plant for an introgressed phenotypic trait.

The presently disclosed subject matter also provides methods forreliably and predictably introgressing yield traits (e.g., starch,protein, oil, and/or ethanol production traits) into maize germplasm. Insome embodiments, the methods comprise using one or more nucleic acidmarkers for marker-assisted selection among maize lines to be used in amaize breeding program, wherein the nucleic acid markers are selectedfrom the group including, but not limited to, SEQ ID NOs: 1-173, andintrogressing the desired trait into the non-trait carrying maizegermplasm. In some embodiments, the one or more nucleic acid markers areselected from the group including, but not limited to, markers forpositive or negative alleles of yield traits (e.g., starch, protein,oil, and/or ethanol production traits). In some embodiments, themarker-assisted selection comprises the use of an analysis techniqueselected from the group including, but not limited to, SNP analysis,RAPD analysis, RFLP analysis, microsatellite analysis, AFLP analysis,TAQMAN® Assay analysis, and ILLUMINA® GOLDENGATE® Genotyping Assayanalysis.

The presently disclosed subject matter also provides methods for theproduction of an inbred maize plant adapted for conferring, in hybridcombination with a suitable second inbred, improved yield traits (e.g.,starch, protein, oil, and/or ethanol production traits). In someembodiments, the methods comprise (a) selecting a first donor parentalline possessing a desired inbred allele for a first yield traitcomprising an ethanol production trait and also possessing an inbredallele for a second yield trait selected from, but not limited to, astarch trait, a protein trait, an oil trait, and/or a second ethanolproduction trait; (b) crossing the first donor parent line with a secondparental line in hybrid combination to produce an F1 generation, andproducing an F2 generation from the F1 generation, wherein the F2generation comprises a segregating plant population; (c) screening oneor more members of the segregating plant population for presence ofdesired chromosomal loci associated with the first yield trait and withthe second yield trait; (d) identifying a plant in the F2 generation, ora selfed and/or double haploid progeny of a plant from the F2generation, that is homozygous for at least the first yield trait atsufficient loci to produce improved ethanol production in hybridcombination; and (e) establishing from the homozygous plant identifiedin step (d) an inbred maize plant adapted for conferring, in hybridcombination with a suitable second inbred, a yield trait. In someembodiments, the methods further comprise screening the plants of theline that is homozygous for improved yield traits at sufficient loci togive improved yield in hybrid combination.

In some embodiments, the detecting of the desired trait comprisesdetecting at least one allelic form of a polymorphic simple sequencerepeat (SSR) or a single nucleotide polymorphism (SNP). In someembodiments, the detecting comprises amplifying the marker locus or aportion of the marker locus and detecting the resulting amplified markeramplicon. In some embodiments, the amplifying comprises: (a) admixing anamplification primer or amplification primer pair with a nucleic acidisolated from the first Zea mays plant or germplasm, wherein the primeror primer pair is complementary or partially complementary to at least aportion of the marker locus, and is capable of initiating DNApolymerization by a DNA polymerase using the maize nucleic acid as atemplate; and (b) extending the primer or primer pair in a DNApolymerization reaction comprising a DNA polymerase and a templatenucleic acid to generate at least one amplicon. In some embodiments, thenucleic acid is selected from DNA and RNA. In some embodiments, the atleast one allele is an SNP allele and the method comprises detecting theSNP using allele specific hybridization (ASH) analysis. In someembodiments, the amplifying comprises employing a polymerase chainreaction (PCR) or ligase chain reaction (LCR) using a nucleic acidisolated from the first maize plant or germplasm as a template in thePCR or LCR.

As used herein, the term “favorable allele” refers to an allele thepresence of which is desirable in a plant in order to achieve a desiredgoal. For example, a favorable allele can be an allele that isassociated with higher or lower yield (e.g., starch, protein, oil,and/or ethanol production), depending on whether higher or lower levelsof these traits is desired under specific circumstances. In someembodiments, a favorable allele is associated with increased ethanolproduction. In some embodiments, a favorable allele is associated withdecreased protein production. In some embodiments, a favorable allele isassociated with increased starch production.

Table 10 (below) discloses exemplary SNPs that are associated withincreases and decreases of various yield traits. In some embodiments,the favorable allele comprises a nucleotide selected from, but notlimited to, (a) an A at nucleotide position 701 of SEQ ID NO: 1; (b) a Gat nucleotide position 498 of SEQ ID NO: 4; (c) a T at nucleotideposition 587 of SEQ ID NO: 7; (d) a G at nucleotide position 708 of SEQID NO: 10; (e) a C at nucleotide position 140 of SEQ ID NO: 13; (f) an Aat nucleotide position 116 of SEQ ID NO: 16; (g) an A at nucleotideposition 269 of SEQ ID NO: 19; (h) an A at nucleotide position 280 ofSEQ ID NO: 22; (i) a T at nucleotide position 374 of SEQ ID NO: 25; (j)a G at nucleotide position 236 of SEQ ID NO: 28; (k) a G at nucleotideposition 605 of SEQ ID NO: 31; (l) a CGA trinucleotide sequence atnucleotide positions 349-351 of SEQ ID NO: 34; (m) a C at nucleotideposition 389 of SEQ ID NO: 37; (n) a G at nucleotide position 66 of SEQID NO: 40; (o) a T at nucleotide position 278 of SEQ ID NO: 43; (p) a Gat nucleotide position 463 of SEQ ID NO: 46; (q) a G at nucleotideposition 510 of SEQ ID NO: 49; (r) a G at nucleotide position 134 of SEQID NO: 52; (s) an A at nucleotide position 367 of SEQ ID NO: 55; (t) a Gat nucleotide position 119 of SEQ ID NO: 58; (u) a G at nucleotideposition 347 of SEQ ID NO: 61; and (v) an A at nucleotide position 356of SEQ ID NO: 64. In some embodiments, the nucleotide present at theaforementioned positions of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25,28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, and 64 is determined ina plant.

Knowledge of the nucleotides present at these positions allows one ofskill to determine whether or not the plant carries an allele ofinterest. If so, the plant can be chosen for further breeding. Thus, oneof ordinary skill in the art would understand that plants can beaffirmatively chosen for further use based on identifying favorablealleles at specific genomic sites (e.g., those listed hereinabove).However, it is also noted that the absence of a favorable allele at aspecific site can also be used to affirmatively reject a certain plantas a candidate for further breeding.

For example, MTL1 is associated with markers that comprise SEQ ID NO: 1and informative fragments thereof. As set forth in Table 10 (below), anA nucleotide at position 701 of SEQ ID NO: 1 is associated withdecreased protein, increased starch, and increased digestibility at 48and 72 hours. Thus, identification of the nucleotide at position 701 ofSEQ ID NO: 1 in a plant can be employed to determine whether or not theplant is likely to carry the decreased protein, increased starch, andincreased digestibility at 48 and 72 hours alleles. If the plant has anA nucleotide at position 701 of SEQ ID NO: 1 in one or both of itschromosome 5s, it is a candidate for further breeding in those instancesin which decreased protein, increased starch, and/or increaseddigestibility at 48 and 72 hours is desirable.

However, as set forth in Table 10, having a G at nucleotide 701 of SEQID NO: 1 would have the opposite effect of having an A at this position.Thus, Table 10 also indicates that if increased protein, decreasedstarch, and/or decreased digestibility at 48 and 72 hours is desired,plants can be screened for what nucleotide is present at position 701 ofSEQ ID NO: 1, and those that have a G at this position in one or both ofits chromosome 5s can be chosen under these circumstances, and thosethat have an A at this position in one or both of its chromosome 5s canbe rejected. Thus, with respect to the alleles disclosed in Table 10,allele 1 or allele 2 can be considered to have opposite effects, andeither can be a favorable allele (affirmatively chosen) or anunfavorable allele (affirmatively rejected) depending on the trait thatis of interest.

In some embodiments, the at least one allele is correlated with at leastone improved yield trait, the method comprising introgressing the allelein the first Zea mays plant or germplasm into a second Zea mays plant orgermplasm to produce an introgressed Zea mays plant or germplasm. Insome embodiments, the second Zea mays plant or germplasm displays moreimproved yield traits as compared to the first Zea mays plant orgermplasm, and wherein the introgressed Zea mays plant or germplasmdisplays an increased improved yield trait as compared to the second Zeamays plant or germplasm.

The presently disclosed subject matter also provides methods forproducing maize plants which carry improved yield traits (e.g., improvedstarch, protein, oil, and/or ethanol production traits). In someembodiments, the methods comprise providing a Zea mays plant whichcontains one or more alleles that confer improved yield, the allelesbeing characterized by one or more sets of loci. These alleles can beemployed individually or in combinations within a set of yield traits(e.g., any of starch, protein, oil, and ethanol production traits) orbetween different sets of yield traits (e.g., starch, protein, oil,and/or ethanol production traits). Alternatively, all sets of yieldtraits can be employed. Combinations of yield traits or sets of yieldtraits selected for increase or decrease of any of a number of thepresently disclosed yield traits can be employed to alter yield, andcombinations of these yield traits can be introgressed into a singleplant (i.e., “stacked”), if desired.

In some embodiments, a marker locus associated with an improved yieldtrait (e.g., a starch, protein, oil, and/or ethanol production trait)displays a genetic recombination frequency of less than about 50%, 25%,20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% with a geneticlocus encoding a yield trait. In some embodiments, the marker locusassociated with an improved yield trait is selected from marker lociassociated with an increased starch trait and associated with adecreased protein trait. In some embodiments, the marker locusassociated with improved yield comprises a plurality of loci selectedfrom marker loci associated with starch traits and ethanol productiontraits. In some embodiments, the marker locus associated with improvedyield is selected from, but not limited to, marker loci localizingwithin the chromosome intervals of 20 cM. In some embodiments, thegermplasm is a Zea mays line or variety.

In some embodiments, the detecting of the desired trait comprisesdetecting at least one allelic form of a polymorphic simple sequencerepeat (SSR) or a single nucleotide polymorphism (SNP). In someembodiments, the detecting comprises amplifying the marker locus or aportion of the marker locus and detecting the resulting amplified markeramplicon. In some embodiments, the amplifying comprises (a) admixing anamplification primer or amplification primer pair with a nucleic acidisolated from the first Zea mays plant or germplasm, wherein the primeror primer pair is complementary or partially complementary to at least aportion of the marker locus and is capable of initiating DNApolymerization by a DNA polymerase using the maize nucleic acid as atemplate; and (b) extending the primer or primer pair in a DNApolymerization reaction comprising a DNA polymerase and a templatenucleic acid to generate at least one amplicon. In some embodiments, thenucleic acid is selected from DNA and RNA. In some embodiments, the atleast one allele is an SNP allele and the methods comprise detecting theSNP using allele specific hybridization (ASH) analysis, TAQMAN® AssayAnalysis (Applied Biosystems, Inc., Foster City, Calif., United Statesof America), and/or the ILLUMINA® GOLDENGATE® Genotyping Assay analysis(Illumina Inc., San Diego, Calif., United States of America). In someembodiments, the amplifying comprises employing a polymerase chainreaction (PCR) or ligase chain reaction (LCR) using a nucleic acidisolated from the first maize plant or germplasm as a template in thePCR or LCR.

In some embodiments, the at least one allele is a favorable allele thatpositively correlates with an improved yield trait (e.g., an improvedstarch, protein, oil, and/or ethanol production trait). In someembodiments, the at least one allele is a favorable allele thatnegatively correlates with an improved yield trait (e.g., a starch,protein, oil, and/or ethanol production trait). In some embodiments, theat least one allele comprises two or more alleles. In some embodiments,the at least one allele is correlated with an improved yield trait(e.g., an starch, protein, oil, and/or ethanol production trait) and themethods comprise introgressing the allele in the first Zea mays plant orgermplasm into a second Zea mays plant or germplasm to produce anintrogressed Zea mays plant or germplasm. In some embodiments, thesecond Zea mays plant or germplasm is characterized by more improvedyield traits as compared to the first Zea mays plant or germplasm, andwherein the introgressed Zea mays plant or germplasm displays anincrease in the number of improved yield traits as compared to thesecond Zea mays plant or germplasm.

A method for introgressing an allele associated with a pre-selectedyield trait into Zea mays germplasm, the method comprising: (a)selecting from a population of Zea mays plants at least one Zea maysplant that comprises an allele of a yield locus associated with apre-selected yield trait, wherein the yield locus is genetically linkedto at least one marker locus that co-segregates with the yieldassociated trait, and further wherein the yield locus comprises anucleotide sequence at least 85% identical to a Zea mays genomicsequence selected from the group including, but not limited to,nucleotides 49337-50164 of GENBANK® Accession No. AC209208.3;nucleotides 43103-43821 of GENBANK® Accession No. AC206616.3;nucleotides 50033-20828 of GENBANK® Accession No. AC204769.3;nucleotides 99595-100453 of GENBANK® Accession No. AC214243.4;nucleotides 56729-57382 of GENBANK® Accession No. AC185458.4;nucleotides 113858-114950 of GENBANK® Accession No. AC211735.3;nucleotides 121592-123221 of GENBANK® Accession No. AC212758.3;nucleotides 159378-160234 of GENBANK® Accession No. AC203779.6;nucleotides 4367-4883 of GENBANK® Accession No. AC196146.3; nucleotides106354-107115 of GENBANK® Accession No. AC214129.2; nucleotides2608-3395 of GENBANK® Accession No. AC183312.5; nucleotides116067-116863 of GENBANK® Accession No. AC194834.3; nucleotides66678-67232 of GENBANK® Accession No. AC197472.2; nucleotides10855-10962 or nucleotides 103384-103629 of GENBANK® Accession No.AC204604.3; nucleotides 173922-174662 of GENBANK® Accession No.AC197469.3; nucleotides 181778-182591 of GENBANK® Accession No.AC210263.4; nucleotides 120187-121112 of GENBANK® Accession No.AC203332.3; nucleotides 85472-86260 of GENBANK® Accession No.AC198211.4; nucleotides 147402-148055 of GENBANK® Accession No.AC191759.3; nucleotides 166218-166660 of GENBANK® Accession No.AC204581.3; and nucleotides 100621-101358 of GENBANK® Accession No.AC212580.4; and (b) introgressing the allele of the yield locusassociated with the pre-selected yield trait into Zea mays germplasmthat lacks the allele.

A method for introgressing an allele associated with a pre-selectedyield trait into Zea mays germplasm, the method comprising (a) selectingfrom a population of Zea mays plants at least one Zea mays plantcomprising at least one allele associated with a pre-selected yieldtrait, wherein the allele comprises a nucleotide sequence selected fromthe group including, but not limited to, SEQ ID NOs: 67-110; and (b)introgressing the allele associated with the pre-selected yield traitinto Zea mays germplasm that lacks the allele.

A method for introgressing an allele associated with a pre-selectedyield trait into Zea mays germplasm, the method comprising (a) selectingfrom a population of Zea mays plants at least one Zea mays plant thatcomprises an allele of a yield locus associated with a pre-selectedyield trait, wherein the yield locus is selected from the groupincluding, but not limited to, MTL1-18, MTPL1-10, MTSL1-6, and MTOL1-3,and further wherein: (1) MTL1, MTPL1, and MTSL1 map to Zea mayschromosome 5 and comprise a nucleotide sequence at least 85% identicalto SEQ ID NO: 1; (2) MTL2 and MTSL2 map to Zea mays chromosome 5 andcomprise a nucleotide sequence at least 85% identical to SEQ ID NO: 4;(3) MTL3 and MTSL3 map to Zea mays chromosome 5 and comprise anucleotide sequence at least 85% identical to SEQ ID NO: 7; (4) MTL4,MTPL2, and MTSL4 map to Zea mays chromosome 3 and comprise a nucleotidesequence at least 85% identical to SEQ ID NO: 10; (5) MTL5 maps to Zeamays chromosome 2 and comprises a nucleotide sequence at least 85%identical to SEQ ID NO: 13; (6) MTL6 maps to Zea mays chromosome 5 andcomprises a nucleotide sequence at least 85% identical to SEQ ID NO: 16;(7) MTL7 and MTPL3 map to Zea mays chromosome 7 and comprise anucleotide sequence at least 85% identical to SEQ ID NO: 19; (8) MTL8and MTPL4 map to Zea mays chromosome 5 and comprise a nucleotidesequence at least 85% identical to SEQ ID NO: 22; (9) MTL9 maps to Zeamays chromosome 7 and comprises a nucleotide sequence at least 85%identical to SEQ ID NO: 25; (10) MTL10 and MTOL3 map to Zea mayschromosome 1 and comprise a nucleotide sequence at least 85% identicalto SEQ ID NO: 28; (11) MTL11 maps to Zea mays chromosome 2 and comprisesa nucleotide sequence at least 85% identical to SEQ ID NO: 31; (12)MTL12 maps to Zea mays chromosome 10 and comprises a nucleotide sequenceat least 85% identical to SEQ ID NO: 34; (13) MTL13 and MTSL5 map to Zeamays chromosome 8 and comprise a nucleotide sequence at least 85%identical to SEQ ID NO: 37; (14) MTL14 and MTPL9 map to Zea mayschromosome 1 and comprise a nucleotide sequence at least 85% identicalto SEQ ID NO: 40; (15) MTL15 maps to Zea mays chromosome 5 and comprisesa nucleotide sequence at least 85% identical to SEQ ID NO: 43; (16)MTL16 maps to Zea mays chromosome 1 and comprises a nucleotide sequenceat least 85% identical to SEQ ID NO: 46; (17) MTL17 and MTPL8 map to Zeamays chromosome 5 and comprise a nucleotide sequence at least 85%identical to SEQ ID NO: 49; (18) MTL18 and MTPL7 map to Zea mayschromosome 10 and comprise a nucleotide sequence at least 85% identicalto SEQ ID NO: 52; (19) MTOL1 maps to Zea mays chromosome 1 and comprisesa nucleotide sequence at least 85% identical to SEQ ID NO: 55; (20)MTPL5 maps to Zea mays chromosome 4 and comprises a nucleotide sequenceat least 85% identical to SEQ ID NO: 58; (21) MTSL6 and MTPL10 map toZea mays chromosome 6 and comprises a nucleotide sequence at least 85%identical to SEQ ID NO: 61; and (22) MTPL6 and MTOL2 map to Zea mayschromosome 7 and comprise a nucleotide sequence at least 85% identicalto SEQ ID NO: 64; and (b) introgressing the allele of the yield locusinto Zea mays germplasm that lacks the allele, whereby an alleleassociated with a pre-selected yield trait is introgressed into Zea maysgermplasm. In some embodiments, the percent identity is over at least25, 50, 75, or 100 nucleotides of the indicated SEQ ID NO. In someembodiments, the percent identity is over the full length of theindicated SEQ ID NO. In some embodiments, the percent identity excludesconsideration of any position at which the indicated SEQ ID NO. includesan “n” nucleotide from the percent identity calculation.

IV. Production of Improved Trait Carrying Maize Plants by TransgenicMethods

The use of SNPs as defined or trait-conferring parts, for producing atrait carrying maize plant, which by introducing a nucleic acid sequencecomprising the trait-associated allele of the SNP into a recipientplant.

A donor plant, with the nucleic acid sequence that comprises ayield/ethanol production trait allele can be transferred to therecipient plant lacking the allele. The nucleic acid sequence can betransferred by crossing an yield/ethanol production trait carrying donorplant with a non-trait carrying recipient plant (i.e., byintrogression), by transformation, by protoplast transformation orfusion, by a doubled haploid technique, by embryo rescue, or by anyother nucleic acid transfer system. Then if desired optionally ofprogeny plants comprising one or more of the presently disclosedyield/ethanol production trait alleles can be selected. A nucleic acidsequence comprising an yield/ethanol production trait allele can beisolated from the donor plant using methods known in the art, and thethis isolated nucleic acid sequence can transform the recipient plant bytransgenic methods. This can occur with a vector, in a gamete, or othersuitable transfer element, such as a ballistic particle coated with thenucleic acid sequence.

Plant transformation generally involves the construction of anexpression vector that will function in plant cells and includes nucleicacid sequence that comprises an allele associated with the yield/ethanolproduction trait, which vector can comprise a yield/ethanol productiontrait-conferring gene. This gene usually is controlled or operativelylinked to one or more regulatory element, such as a promoter. Theexpression vector can contain one or more such operably linkedgene/regulatory element combinations, provided that at least one of thegenes contained in the combinations encodes yield/ethanol productiontrait. The vector(s) can be in the form of a plasmid, and can be used,alone or in combination with other plasmids, to provide transgenicplants that are better yield/ethanol production plants, usingtransformation methods known in the art, such as the Agrobacteriumtransformation system.

Transformed cells often contain a marker allow transformationidentification. The marker is adapted to be recovered by negativeselection (by inhibiting the growth of cells that do not contain theselectable marker gene), or by positive selection (by screening for theproduct encoded by the marker gene). Many commonly used selectablemarker genes for plant transformation are known in the art, and include,for example, genes that code for enzymes that metabolically detoxify aselective chemical agent that can be an antibiotic or a herbicide, orgenes that encode an altered target which is insensitive to theinhibitor. Several positive selection methods are known in the art, suchas mannose selection. Alternatively, marker-less transformation can beused to obtain plants without the aforementioned marker genes, thetechniques for which are also known in the art.

V. Improved Plants, and Plant Parts, Seeds, Tissue Cultures, and BiomassDerived Therefrom

The presently disclosed subject matter also provides improved maizeplants, parts, seeds, progeny, and tissue cultures produced by any ofthe presently disclosed methods.

In some embodiments, the presently disclosed subject matter providesimproved maize plants or a part, seed, progeny, and/or tissue culturethereof, which evidences a selected yield trait (e.g., a starch,protein, oil, and/or ethanol production trait), a genome homozygous withrespect to one or more genetic alleles which are present in a firstparent and not present in a second parent of the improved maize plant,in some embodiments, (a) the second parent evidences a more improvedyield trait (e.g., a more improved starch, protein, oil, and/or ethanolproduction trait) than the first parent; and (b) the improved plantcomprises one or more alleles from the first parent that evidence animproved yield trait in hybrid combination in at least one locusselected from (i) a starch locus with a desired starch allele; (ii) aprotein locus with a desired protein allele; (iii) a digestibility locuswith a desired ethanol production allele; and/or (iv) an oil locus witha desired oil allele; and the desired trait is not significantly lessthan that of the first parent in the same hybrid combination and yieldcharacteristics which are not significantly different than those of thesecond parent in the same hybrid combination.

In some embodiments, the improved maize plants comprise each of a starchlocus and a digestibility locus, and have improved yield trait whencompared to a substantially identical maize plant not comprising theloci. In some embodiments, the improved maize plants comprise each of astarch locus with an allele for increased starch and a protein locuswith an allele for decreased protein, and which have improved yieldtraits when compared to a substantially identical maize plant notcomprising the loci.

In some embodiments, the improved maize plants, or parts, seeds,progeny, and tissue cultures thereof, comprise progeny of a crossbetween first and second inbred or hybrid lines, wherein one or morealleles conferring selected yield traits (e.g., starch, protein, oil,and/or ethanol production traits) are present in a homozygous state inthe genome of one or the other or both of the first and second inbred orhybrid lines, such that the genome of the first and second inbreds orhybrids together donate to the improved maize plant or part thereof acomplement of alleles sufficient to confer the improved yield trait(s).The presently disclosed subject matter also provides hybrids, or a partthereof, formed with the presently disclosed improved maize plants.

The presently disclosed subject matter also provides maize plants, orparts, seeds, and tissue cultures thereof, formed by selfing thepresently disclosed improved yield traited hybrid(s).

The presently disclosed subject matter also provides maize plants, orparts, seeds, progeny, and tissue cultures thereof, that have one ormore desired yield traits produced by the presently disclosed methods.In some embodiments, the maize plants that have one or more improvedyield traits are hybrid maize. The presently disclosed subject matteralso provides biomass and seed produced by the presently disclosed maizeplants.

As such, the presently disclosed subject matter provides improved maizeplants, or parts, seeds, progeny, tissue cultures, and biomass derivedthereof, which evidence a selected yield trait, optionally wherein thegenome of the improved maize plant, or the part, seed, progeny, ortissue culture thereof is homozygous with respect to one or more geneticalleles associated with the selected trait.

In some embodiments, the improved maize plant, or the part, seed,progeny, or tissue culture thereof comprises a genome that is homozygouswith respect to one or more genetic alleles that are present in a firstparent and not present in a second parent of the improved maize plant.In some embodiments, the second parent evidences more improved yieldtraits than the first parent, and the improved plant comprises one ormore alleles from the first parent that evidence improved yield traitsin hybrid combination in at least one locus selected from, but notlimited to, a starch locus with a desired starch allele, a protein locuswith a desired protein allele, a digestibility locus with a desiredprotein allele, and/or an oil locus with a desired oil allele, and thedesired trait is not significantly less than that of the first parent inthe same hybrid combination and yield characteristics which are notsignificantly different than those of the second parent in the samehybrid combination.

In some embodiments, the improved maize plants comprise each of a starchlocus and a digestibility (i.e., ethanol production) locus, and haveimproved yield traits when compared to a substantially identical maizeplant not comprising the starch locus and the digestibility locus. Insome embodiments, the improved maize plants comprise each of a starchlocus with an allele for increased starch and a protein locus with anallele for decreased protein, and which have improved yield traits whencompared to a substantially identical maize plant not comprising thestarch locus and the protein locus.

In some embodiments, the improved maize plants or parts thereof compriseprogeny of a cross between first and second inbred or hybrid lines,wherein one or more alleles conferring selected yield traits are presentin a homozygous state in the genome of one or the other or both of thefirst and second inbred or hybrid lines, such that the genome of thefirst and second inbreds or hybrids together donate to the improvedmaize plant or part thereof a complement of alleles sufficient to conferthe improved yield trait(s). The presently disclosed subject matter alsoprovides hybrids, or a part thereof, formed with the presently disclosedimproved maize plants.

The presently disclosed subject matter also provides maize plants, or apart thereof, formed by selfing the presently disclosed improved yieldtraited hybrids.

The presently disclosed subject matter also provides maize plants thathave desired yield traits occurring in maize produced by the presentlydisclosed methods. In some embodiments, the maize plants that haveimproved yield traits are hybrid maize. The presently disclosed subjectmatter also provides biomass and seed produced by the presentlydisclosed maize plants.

The presently disclosed subject matter also provides Zea mays plantshaving one or more improved yield traits associated with the presence ofMTL1-18, MTPL 1-9, MTSL 1-6, and/or MTOL 1-3 as defined herein in ahomozygous genetic background.

The presently disclosed subject matter also provides maize plants thathave one or more yield traits, wherein the plant is a plant of thespecies Zea mays, and the plant comprises at least one chromosomalregion that confers enhanced or decreased ethanol production traits fromthe chromosome group comprising chromosomes 1, 2, 3, 5, 7, 8 and 10 andfurther wherein the at least one chromosomal region that confers anincrease or decrease in ethanol production which is linked to at leastone marker selected from markers associated with one of MTL1-18.

The presently disclosed subject matter also provides maize plants thathave one or more yield traits, wherein the plant is a plant of thespecies Zea mays, and the plant comprises at least one chromosomalregion that confers decreased or increased protein production traitsfrom the chromosome group comprising chromosomes 1, 3, 5, 7, and 10 andfurther wherein the at least one chromosomal region that confers anincrease or decrease in protein production which is linked to at leastone marker selected from markers associated with one of MTPL1-10.

The presently disclosed subject matter also provides maize plants thathave one or more yield traits, wherein the plant is a plant of thespecies Zea mays, and the plant comprises at least one chromosomalregion that confers enhanced or decreased starch traits from thechromosome group comprising chromosomes 3, 5, 6, 8, and further whereinthe at least one chromosomal region that confers an increase or decreasein starch which is linked to at least one marker selected from markersassociated with one of MTSL1-6.

The presently disclosed subject matter also provides maize plants thathave one or more yield traits, wherein the plant is a plant of thespecies Zea mays, and the plant comprises at least one chromosomalregion that confers enhanced or decreased oil traits from the chromosomegroup comprising chromosomes 1 and 7, and further wherein the at leastone chromosomal region that confers an increase or decrease in oil whichis linked to at least one marker selected from markers associated withone of MTOL1-3.

The presently disclosed subject matter also provides parts of the plantsdefined herein. In some embodiments, the plant part is pollen, ovule,leaf, embryo, root, root tip, anther, flower, fruit, stem, shoot, seed;cell, rootstock, protoplast, or callus.

EXAMPLES

The following Examples provide illustrative embodiments. In light of thepresent disclosure and the general level of skill in the art, those ofskill will appreciate that the following Examples are intended to beexemplary only and that numerous changes, modifications, and alterationscan be employed without departing from the scope of the presentlydisclosed subject matter.

Example 1 Initial Plant Materials—Platform and Panel

PLATFORM DEVELOPMENT. A group of 2998 temperate, tropical, andsubtropical corn inbreds were selected from substantially homozygouscorn inbred material. This group was reduced to 2075 inbreds byelimination of genetically modified corn, and low seed availability ofcertain corn seed. This was referred to as the inbred “platform”. Thisplatform was used for association marker work and phenotypic evaluation.

PANEL DEVELOPMENT. The platform group of corn inbred material wasanalyzed and further selected based on flowering data and furthergrouped by growing degree units (GDU). 1201 lines were characterized bygrowing degree units and grouped as follows.

-   -   A (1270-1390)=279 Lines    -   B (1391-1510)=700 Lines    -   C (1511-1630)=222 Lines    -   Total=1201 Lines

The 1201 lines were then filtered for allelic diversity with markers.The results eliminated the very related lines (e.g., Neis similaritygreater than or equal to 0.9 were eliminated). The diverse groupresulted in the selection of an inbred panel of 600 lines categorizedinto their flower data based growing degree unit groups.

-   -   A (1270-1390)=180 Lines    -   B (1391-1510)=243 Lines    -   C (1511-1630)=177 Lines    -   Total=600 Lines

The inbred panel and inbred platform were used for assaying associationmarkers and for phenotypic evaluation.

Example 2 Phenotypic Data

The inbred platform and the inbred panel were each evaluated for eightphenotypic traits: moisture, density, oil, starch, protein, anddigestibility (i.e., ethanol production) at 24, 48, and 72 hours asshown in Table 4. Moisture was the grain moisture after air drying.Density was recorded as a measure of kernel density. Oil, protein, andstarch were each measured by well known methods in the industry and wererecorded as a percentage.

Phenotypic information for inbred lines (1765 entries) was determined.The phenotypic information for certain traits corresponded to acalibration of the Near Infrared Spectroscope (NIR) machine. The NIRprovided information on the yield traits starch, protein, oil, moisture,and density. Later, phenotypic information also included NIR phenotypicdata for ethanol production traits (e.g., digestibility 24,digestibility 48, and digestibility 72). The phenotypic information ofthose lines that were part of the inbred platform (1732 inbreds) wasextracted for the analysis. The results are shown in Tables 5 and 6.

TABLE 4 Traits and Trait Definitions SPIRIT Trait Trait Code SPIRITTrait Definition Moisture GMADP Grain moisture after air drying OilOIL_P Oil % Density DEN_N Kernel density Starch STC_P Grain starchcontent % Protein PRTNP Protein % Digestibility 24 DG24P Dry grindethanol % after 24 hours fermentation Digestibility 48 DG48P Dry grindethanol % after 48 hours fermentation Digestibility 72 DG72P Dry grindethanol % after 72 hours fermentation

TABLE 5 Inbred Platform (1753 Lines Phenotyped) Std Mean Trait Mean DevStd Err Min Max N Moisture 10.64 0.69 0.02 9.04 15.81 1753 Oil 3.93 0.630.02 2.36 6.87 1753 Density 1.47 0.04 0.00 1.34 1.61 1753 Starch 70.012.35 0.06 59.87 78.01 1732 Protein 12.46 1.46 0.04 8.04 17.75 1728 DG24P5.01 0.91 0.02 1.83 8.21 1732 DG48P 6.33 1.09 0.03 1.45 10.36 1732 DG72P7.86 1.02 0.02 2.49 11.56 1732

TABLE 6 Inbred Panel (576 Lines Phenotyped) Std Mean Trait Mean Dev StdErr Min Max N Moisture 10.67 0.68 0.03 9.04 15.26 569 Oil 3.91 0.60 0.032.58 6.10 569 Density 1.47 0.04 0.00 1.35 1.61 569 Starch 70.09 2.400.10 62.14 78.01 576 Protein 12.41 1.46 0.06 8.04 16.71 574 DGST 24 5.050.93 0.04 1.83 8.21 576 DGST 48 6.39 1.07 0.04 3.42 9.67 576 DGST 727.93 0.99 0.04 4.22 11.56 576

A scatter plot correlation of the yield associated traits from theinbred platform is shown in FIG. 1.

Example 3 Association Mapping of Platform and Panel

The inbred platform and inbred panel were characterized by substantialallelic diversity for the assayed traits. This variability or diversitywas employed in the associated mapping experiments. The associatedmapping used polymorphic SNP markers and controlled the populationstructure of these sets of inbreds. The platform contained at least 5maize heteroic patterns including stiff stalk, non-stiff stalk, iodent,and mixtures of heteroic patterns. An inbred platform of 1732 lines andan inbred panel of 600 of these same lines were used to identifysignificant marker trait associations for traits of interest for yield.

The inbred panel and the inbred platform were phenotyped for traits ofinterest. Linear models were applied to identify marker traitassociations (MTAs) for eight traits (starch, protein, moisture,density, oil, digestibility 24, digestibility 48, and digestibility 72)and 1654 SNP markers. Principal Components were included as covariatesin the models to reduce the false positive rates and increase thecoefficient of determination (R²) of the models by including the mostexplicative variables into the model. A total of 122 MTAs weresignificant in General Linear Models (GLM) at an experiment-wisesignificance level of 5%. All the 122 were also significant (p-value<0.05) in Mixed Linear Models (MLM) that included Kinship estimates asthe additive relationship matrix to further dissect the geneticrelatedness among the inbred lines. Nine of these associations werecommon to more than one trait and their allelic effects on the traitswere as expected (e.g., positive correlation between the traits starchand ethanol production and negative correlation between the traitsprotein and starch).

Genotypic Data

TAQMAN® SNPs.

Genotypic information was extracted for 2052 inbred lines that wereincluded in the association platform list. A total of 496 SNPs were usedfor association and population structure analysis.

Illumina SNPs.

The Illumina Plex1 (version 1) was composed of 1536 SNPs. For the inbredpanel, 559 lines were submitted for genotyping from which data wasreceived for 485 lines (Table 7).

TABLE 7 Summary of Illumina PLEX1 Genotyping Results Inbred # LinesSubmitted to Data from Panel Panel PLEX 1 PLEX1 % Inbred 600 559 48580.83% SS 599 566 516 86.14% NSS 600 583 529 88.17% Total 1228 1174 106886.97%

Linear models were applied to identify marker trait associations (MTAs)for these traits and 1654 SNP markers.

Genotypic data from the Plex1 was converted to a matrix form and rawdata from 1219 SNP was analyzed using PowerMarker software (Liu & Muse,2005) and 61 monomorphic markers were removed.

Kinship Analysis

Kinship analysis was initially done using genotypic data of 496 TAQMAN®SNP assays. Four different approaches for the estimation of Kinship (orco-ancestry coefficients) were compared: Kinship as the proportion ofshared alleles, Kinship according to J. Nason (described in Loiselle etal., 1995; calculated in SPAGeDi; Hardy & Vekemans, 2002), Kinshipaccording to Ritland (1996; calculated in SPAGeDi), and Kinship ascalculated in TASSEL 2.0.1. All pairwise comparisons were highlysignificant (p<0.0001). Proportion of shared alleles was employed. Ithas been suggested that the robustness in the estimation of Kinship canbe affected by the number of markers. In order to test how different theKinship matrices might be with different sets of markers, Kinship(pShared) was calculated with the 1158 SNPs of the Illumina Plex1. The Kmatrix obtained with the Plex1 SNPs was used in the mixed models for theinbred panel (see Table 8).

TABLE 8 Mantel Test Comparison of Kinship Matrices Calculated with 496TAQMAN ® SNPs and 1158 PLEX1 SNPs Distance A Distance B Correlation pValue K matrix K matrix 0.8750 0.0000 (Kpshared) 496 (Kpshared) 1158TAQMAN ® SNPs ILLUMINA ® SNPs

PCA Analysis

Principal Component Analysis (PCA) or “eigenanalysis” has been proposedas an alternative to Structure software (Pritchard et al., 2000) forinferring population structure from genotypic data (Patterson et al.,2006). PCA has some advantages over Structure such as the processingspeed for large datasets and avoiding the need of selecting a specificnumber of sub-populations. PCA was performed using the software SMARTPCAthat is part of Eigenstrat using data from the Illumina PLEX1.

Selection of PCs Based on Association with the Trait of Interest.

The utilization of Principal Components (PCs) as covariates in linearmodel-based association mapping has relied in the assumption that thefirst PC's are the best covariates because the explain most of thegenetic variation found with the markers (Zhao et al., 2007). Thecorrelation between PCs and the phenotype was dependent on the trait andsometimes large PCs did not explain much of the variation whereas minorPC's explained a considerable percentage of the variation for certaintraits. Both GLM and MLM were employed to assess the significance ofeach of 50 PCs and to estimate the percentage of the variation explainedby them.

TASSEL.

The java-based software TASSEL (Trait Analysis by aSSociation, Evolutionand Linkage) incorporates linear models (both general and mixed)approaches to establish association between markers and phenotypes whilecontrolling for population and family structure (Bradbury et al., 2007).Population structure (Q) and/or Kinship (K) estimates can beincorporated in the models to reduce the number of false positives. Itis also possible to replace the Q (Structure) matrix by a PCA matrix(Eigenvalues; Price et al., 2006; Zhao et al., 2007).

Association Models in TASSEL.

The different models used in TASSEL are shown in the Table 9.

TABLE 9 Models used for association in TASSEL General Lineal ModelsMixed Lineal Models 1) Phenotype = Marker + 2) Phenotype = Marker + KselectedPCs (Eigenvalues) (pshared) + selectedPCs (Eigenvalues)

Adjustments for Multiple Testing.

The GLM procedure in TASSEL included the option to perform permutationsto find out the experiment-wise error rate that corrected foraccumulation of false positives when doing multiple comparisons. A totalof 1,000 permutations were used. The MLM procedure did not includecorrection for multiple testing. In addition, the software QVALUE(Storey, 2002) was used to calculate q-values to control for the falsediscovery rate (FDR). The q-values were similar to p-values since theygave each hypothesis test a measure of significance in terms of acertain error rate. The q-values were useful for assigning a measure ofsignificance to each of many tests performed simultaneously.

Association Results in Inbred Platform.

Phenotypic data was available for 1732 lines out of the 2052 lines ofthe inbred platform with marker information in the TAQMAN® 496 SNP set.The use of Mixed Linear Models (MLMs) to detect marker traitassociations in data sets of considerable size (>1000) was limited bythe computation time required to analyze the Kinship component of themodel (i.e., it takes TASSEL 2.0.1 240 computing hours to finalize a K+Qmodel for one trait with 1732 lines and 488 SNPs when running at adesignated 1536 Mb of RAM in a Core2 Duo PC). As an alternative,refining the General Linear Models to correct for population structureas much as possible without the need of the Kinship matrix wasattempted.

Comparison between several GL models showed that the selection of PCsbased on trait significance helped to reduce the bias towardssignificance. The comparison also showed that the grouping in k=10subpopulations according to STRUCTURE results gave skewed resultstowards significance.

The data from TASSEL 2.1 of the GLM for both the 496 TAQMAN® SNPs forthe 8 traits and the LSmeans per locus was run. The selection of thesignificant PCs as covariates in the linear models helped to control thedistribution of p-values (i.e. avoid large numbers of false positives).However, variation was observed between the different traits.

A total of 85 SNPs showed experiment-wise p-values of less than 0.05 inthe GLM (Trait=marker+selected PCs) in the inbred platform. The traitswith the highest number of significant marker trait associations (MTAs)were oil and protein with 13 and the one with least was moisture with 7.A total of 15 SNPs with significant p-values (experiment-wise p-value<5%) showed association with more than one trait (see Table 10). In mostcases, the alleles that increased the level of protein decreaseddigestibility and/or starch (see e.g. the traits associated with SEQ IDNOs: 1, 4, 7, and 10).

TABLE 10 Loci with Experiment-Wise p-Values of Less than 0.05, TheirDistribution among Traits, and the Effects of Reference Alleles on theDifferent Traits SEQ ID Allele Allele NO: Pos 1 2 Effect of Allele 1 1701 A G Protein (−); Starch (+); DGST 48 (+); DGST 72 (+) 4 498 G AStarch (+); DGST 48 (+); DGST 72 (+) 7 587 T C Starch (+); DGST 48 (+);DGST 72 (+) 10 708 G A Protein (−); Starch (+); DGST 48 (+) 13 140 C TDGST 24 (+); DGST 48 (+) 16 116 A G DGST 24 (+); DGST 48 (+); DGST 72(+) 19 269 A G Protein (−); DGST 24 (+) 22 280 A G Protein (−); DGST 48(+) 25 374 T G Density (+); DGST 24 (−) 28 236 G A Oil (−); DGST 48 (+)31 605 G A DGST 48 (+); DGST 72 (+) 34 349 C T Moisture (−); DGST 24 (+)37 389 C T Starch (+), DGST 72 (+) 40 66 G A Protein (−), DGST 24 (+) 43278 T C DGST 48 (+), DGST 72 (+) 46 463 G A Density (−), DGST 24 (+),DGST 48(+) 49 510 G A Protein (−), DGST 48(+), DGST 72(+) 52 134 G TProtein (−), DGST 48(+), DGST 72(+) 55 367 A G Moisture (−), Oil (−) 58119 G C Density (−); Protein (−) 61 347 G T Starch(+), Protein (−) 64356 A G Oil (+); Protein (−) (−): Allele 1 results in a lower level thanAllele 2; (+): Allele 1 results in a higher level than Allele 2.

With the exception of the SNPs at nucleotide position 140 of SEQ ID NO:13 and nucleotide position 349 of SEQ ID NO: 34, the SNPs identifiedreflected single nucleotide differences that were limited to the statedpositions. Thus, for example, the only difference between a favorableallele and an unfavorable allele (depending on the desired trait) forSEQ ID NO: 1 was whether the nucleotide at position 701 was an A or a G.It was noted, however, that SEQ ID NOs: 13 and 34 were more complex.Nucleotide position 140 of SEQ ID NO: 13, for example, is a C or a T,but review of the sequence surrounding this position in the variousnucleic acid samples indicated that the C derived not from a nucleotidesubstitution but from a deletion. Specifically, in Allele 1 of Table 10for SEQ ID NO: 13, the C was present at position 140 because one of thestring of five Ts shown in positions 136-140 of SEQ ID NO: 13 wasdeleted, which moved the C that followed this string of Ts from position141 in those embodiments of SEQ ID NO: 13 where five Ts were present toposition 140 in those embodiments where only four Ts were present.

Similarly, the SNP that was found in SEQ ID NO: 34 also involved adeletion, in this case a three nucleotide deletion of the CGA sequenceat positions 349-351 of SEQ ID NO: 34. Thus, Allele 1 of SEQ ID NO: 34of Table 10 included the CGA trinucleotide, and thus had a C at position349. Allele 2, on the other hand, had a deletion of the CGAtrinucleotide, resulting in the T that is present at position 352 of SEQID NO: 4 being moved to position 349.

Association Results in Inbred Panel.

Phenotypic data was available for 576 out of the 600 lines thatconstituted the inbred panel. Information from a total of 1654 SNPs wasavailable for the inbred panel. In addition to a larger number of SNPdata, the reduced size of the inbred panel in comparison to the inbredplatform allows to reduce the running time of the Mixed Linear Models.

The data was developed from TASSEL of the two models (GLM and MLM)selected for both the 496 TAQMAN® SNPs and the Illumina PLEX1, and theLSmeans per locus (from the GLM model). The selection of the significantPCs as covariates in the linear models helped to control thedistribution of p-values (i.e. avoid large numbers of false positives).The inclusion of the Kinship matrix as the additive relationship matrixin the mixed model helped to reduce the false positive rate to expectedlevels and to increase the R2 of the models. Mean R2 values for the GLMand MLM (Trait=marker+selectedPCs+Kpshared) for the assayed traits arepresented in Table 11.

TABLE 11 Mean R2 Values for the GLM and MLM for the Assayed Traits MeanR2 Model TRAIT GLM MLM Density 0.15 0.20 Moisture 0.21 0.27 Oil 0.190.25 Protein 0.28 0.32 Starch 0.24 0.28 Digestion 24 0.34 0.38 Digestion48 0.25 0.30 Digestion 72 0.24 0.28

The correlation analysis between the p-values for the different traitssuggested that there were significant correlations for several traitpairs. This correlation pattern followed the phenotypic correlations inwhich, for example, Starch and Protein were correlated withDigestibility at 24, 48, and 72 hours.

A total of nine SNPs with significant p-values (experiment-wise p-value<0.05) showed association with more than one trait (Table 12). Forexample, the loci that correspond to SEQ ID NOs: 49 and 52 showedassociation with Protein, Digestibility 24, and Digestibility 48. Inboth cases the allele that increases the level of protein decreases theyield of ethanol after both 48 and 72 hours of digestion. Only one MTAwas common to Starch and Digestibility (SEQ ID NO: 37) for which, asexpected, an increase in Starch represented an increase in ethanolproduction.

TABLE 12 Loci with Experiment-wise p-value < 0.05 SNP Posi- AlleleAllele LOCUS tion 1 2 Effect of Allele 1 MTL6 116 A G DGST 48 (+), DGST72 (+) (SEQ ID NO: 16) MTL13/MTSL5 389 C T Starch (+), DGST 72 (+) (SEQID NO: 37) MTL14/MTPL9 66 G A Protein (−), DGST 24 (+) (SEQ ID NO: 40)MTL15 278 T C DGST 48 (+), DGST 72 (+) (SEQ ID NO: 43) MTL16 463 G ADensity (−), DGST 24 (+), (SEQ ID NO: 46) DGST 48(+) MTL17/MTPL7 510 G AProtein (−), DGST 48(+), (SEQ ID NO: 49) DGST 72(+) MTL18/MTPL7 134 G TProtein (−), DGST 48(+), (SEQ ID NO: 52) DGST 72(+) MTOL1 367 A GMoisture (−), Oil (−) (SEQ ID NO: 55) MTSL6/MTPL10 347 G T Starch(+),Protein (−) (SEQ ID NO: 61)

By way of example, Table 12 indicates that MTL6, which is a locus thatis associated with SEQ ID NO: 16 and that includes an SNP at position116 of SEQ ID NO: 16. When the locus includes an A nucleotide at thisposition, digestibility (ethanol production) is increased at both 48 and72 hours relative to when a G (or, for that matter, a C or T) is presentat this position. Similarly, MTL15 is a locus associated with SEQ ID NO:43 and that includes an SNP at position 278 of SEQ ID NO: 43. When thelocus includes a T nucleotide at this position, digestibility (ethanolproduction) is increased at both 48 and 72 hours relative to when a C(or, for that matter, a G or T) is present at this position.Additionally, MTL17/MTPL7 is a locus associated with SEQ ID NO: 49 andthat includes an SNP at position 510 of SEQ ID NO: 49. When the locusincludes a G nucleotide at this position, digestibility (ethanolproduction) is increased at both 48 and 72 hours and protein content isdecreased relative to when an A (or, for that matter, a C or T) ispresent at this position.

Example 4 Running Assays to Select for Traits

Each of the lines was phenotyped for each of the traits. The assaysusing the primers to produce amplicons from which the probes could beemployed to detect the allele that was present in each of these lines.The lines which had positive alleles for yield were selected and used inthe next breeding generation. This marker associated selection wasadapted to enhance the yield traits in the next generation of maizegermplasm. The landraces and the families of these lines were different.

The lines employed in the inbred panel and inbred platform disclosedherein represented considerable genetic diversity. A sampling of thelines in the inbred panel and the inbred platform is depicted in FIG. 2.From this sampling of the lines employed, it can be seen that variousdifferent heterotic groups were employed, demonstrating that thevalidations disclosed herein were performed over several differentheterotic groups and/or germplasm pools.

Discussion of the Examples

Correlation of Results of Platform and Panel.

A platform of inbreds selected for diversity (approximately 1201 inbredlines) were analyzed and selected based on flowering data and grouped bygrowing degree units (GDU).

These lines were then filtered for allelic diversity with SNP genomewide sets of markers and related lines were identified and eliminated. Asecond group from the platform was selected based on being diverse. Theselection resulted in an inbred panel of 600 lines, which then were alsocategorized into their flower data and grouped according to growingdegree unit. The inbred platform and the inbred panel were subjected tophenotypic evaluation. The grain moisture, density, oil, protein,starch, and digestibility for ethanol at 24, 48 and 72 hours were takenand recorded. This phenotypic data was then correlated with the plantsgenotypic data based on SNPs to identity the marker trait associationswith the listed traits.

Marker-assisted selection (MAS) of maize germplasm has become a commonpractice in breeding. The efficiency of MAS, however, depends on theaccuracy in detection of markers closely linked to MTLs. Associationmapping has been widely used as an alternative to linkage mapping indetecting MTLs. Association mapping takes advantage of recombinationevents accumulated over many generations, and thus offers potentiallymuch higher mapping resolution than conventional linkage mapping, whichis usually based on biparental crosses.

Effective association mapping in maize relies on the LD structure—sizesand distributions of LD blocks and LD strengths in the blocks on thegenome. LD structure has two implications on association mapping: 1) itdetermines the feasibility of association mapping; 2) it determines thepotential mapping resolution. This approach is based on linkagedisequilibrium (LD) between linked loci. Because LD usually exists onlyin much narrower chromosomal regions, mapped MTLs are mapped with ahigher resolution than linkage mapping. However, LD can occur betweenunlinked loci, which can be undesirable, and spurious LD can be causedby population structure, genotyping errors, etc. As a result, toreliably detect true LD between closed linked loci, sophisticatedstatistical approaches are needed to minimize false positives of variouskinds.

TASSEL, developed by Buckler's lab at Cornell University (Bradbury etal. 2007), is one of the software packages that can achieve this goal.TASSEL is based on mixed linear model with population structure andgenetic correlations being explicitly controlled in the models. Thispackage was employed for association analysis with the data in theseexperiments.

The inbred platform and inbred panel had allelic diversity for the 8traits. This variability or diversity was employed in the associatedmapping experiments. The associated mapping used polymorphic SNP markersand controlled the population structure of these sets of inbreds. Theplatform contained at least 5 maize heteroic patterns including: stiffstalk, nonstiff stalk, iodent, and mixtures of heteroic patterns. Aninbred platform of 1732 (˜600 same as panel) lines/and an inbred panelof 600 of these same lines was used to identify significant marker traitassociations for traits of interest for yield and ethanol. Linear Modelsidentified the associations (MTLs) for eight traits with 1654 SNPmarkers. The models included Principal Components to reduce falsepositives. The MLM also included a Kinship estimate to account forgenetic relatedness. The allelic effects on the traits were determined.These effects correlated with the phenotypic effects noted.

By comparing the results between the inbred panel and the inbredplatform the significant 496 TAQMAN® SNPs in both data sets for the sametrait or for highly correlated traits were identified.

The significant MTAs obtained in the current analysis should beconsidered as MTLs i.e. that they represent a region of the maizegenome. A score system was adapted to rank the MTAs, the SNP assaysand/or the SNP locus. This score system weighs the importance of thetrait (40%), the significance in the other data set (30%) and thesignificance in multiple traits (30%). The total number of points that aMTA accumulate can be used to create priority lists.

Summary of Yield Associated Loci.

SNP assays were performed and contributions to variability weredetermined for the twenty-two loci represented by SEQ ID NOs: 1-173. Theresults are summarized as follows:

SEQ ID NO: 1:

Two SNP assays were included in the analysis. In the inbred platform,the SNP assay was associated with the yield traits Protein (GLMp-adj=0.001), explaining 0.76% of the variation; Starch (GLMp-adj=0.001), explaining 0.53% of the variation; DGST 48 (GLMp-adj=0.005) explaining 0.47% of the variation; and DGST 72 (GLMp-adj=0.001), explaining 0.54% of the variation. In the inbred panel,the SNP assay was significantly associated with the trait DGST 48 (GLMp-adj=0.017; MLM p=0.013), explaining ˜1.2% of the variation (R2=GLM1.36%; MLM 1%). The SNP marker is located on chromosome 5 close to theloci associated with SEQ ID NOs: 16 and 49 (see below). There are NAMQTLs in this region of chromosome 5 for the yield traits Starch andProtein.

SEQ ID NOs: 4 and 7:

Four SNP assays were included in the analysis. In the inbred platform,one SNP assay (located between SEQ ID NOs: 5 and 6) was significantlyassociated with the yield traits Starch (GLM p-adj=0.001), explaining0.55% of the variation; DGST 48 (GLM p-adj=0.001), explaining 0.51% ofthe variation; and DGST 72 (GLM p-adj=0.005) explaining 0.41% of thevariation. A second SNP assay (located between SEQ ID NOs: 8 and 9) wasassociated with the same traits: Starch (GLM p-adj=0.001), explaining0.53% of the variation; DGST 48 (GLM p-adj=0.03), explaining 0.39% ofthe variation; and DGST 72 (GLM p-adj=0.001), explaining 0.57% of thevariation. The SNP markers are located on chromosome 5. There is anoverlapping NSS metaQTL for the yield trait Starch that explains anaverage of 1.2% of the variation observed for the trait.

SEQ ID NO: 10:

Three SNP assays were included in the analysis. In the inbred platform,one SNP assay was significantly associated with the yield traits Protein(GLM p-adj=0.001), explaining 0.71% of the observed variation; Starch(GLM p-adj=0.001), explaining 0.54% of the observed variation; and DGST48 (GLM p-adj=0.001), explaining 0.52% of the observed variation. TheSNP is located on chromosome 3.

SEQ ID NO: 13:

Four SNP assays were included in the analysis. In the inbred Platform,one SNP assay was statistically significant for the yield traits DGST 24(GLM p-adj=9.9E-4), explaining 0.51% of the variation observed; and DGST48 (GLM p-adj=0.048), explaining 0.37% of the variation observed. TheSNP assay was also found to be statistically associated with the yieldtrait Starch in the Inbred Panel (GLM p-adj=0.013; MLM p=0.004),explaining ˜1.35% of the variation observed (R2=GLM 1.43%; MLM 1.32%).The SNP is located on chromosome 2. There is a metaQTL for the traitDGST 72 located on chromosome 2.

SEQ ID NO: 16:

Three SNP assays were included in the analysis. In the inbred platform,one SNP assay was statistically significant for the yield traits DGST 24(GLM p-adj=0.034), explaining 0.37% of the variation observed; and DGST48 (GLM p-adj=9.9E-4), explaining 0.54% of the variation observed. Inthe inbred panel, this same SNP assay was statistically significant forthe yield traits DGST 48 (GLM p-adj=9.9E-4; MLM p=5.9E-4), explaining˜2% of the observed variation (R2=GLM 2.23%; MLM 1.91%); and DGST 72(GLM p-adj=0.002; MLM p=0.0017), explaining ˜1.7% of the observedvariation (R2=GLM 1.77%; MLM 1.64%). The SNP is located on chromosome 5close to the loci associated with SEQ ID NO: 1 and SEQ ID NO: 49 thatare also significant for ethanol-related traits. There are NAM QTLs inthis region of chromosome 5 for the yield traits Starch and Protein.

SEQ ID NO: 19:

Four SNP assays were included in the analysis. In the inbred platform,one SNP assay was associated with the yield traits DGST 24 (GLMp-adj=0.019), explaining 0.4% of the observed variation; and Protein(GLM p-adj=0.043), explaining 0.41% of the observed variation. In theinbred panel, this SNP assay was statistically significant for the yieldtrait Oil (GLM p-adj=9.9E-4; MLM p=0.0016) explaining ˜1.9% of thevariation observed (R2=GLM 2.05; MLM 1.72). The SNP marker is located onchromosome 7 close to the locus associated with SEQ ID NO: 64 that wassignificantly associated with Protein and Oil.

SEQ ID NO: 22:

Two SNP assays were included in the analysis. In the inbred platform,one SNP assay was significantly associated with the yield traits Protein

(GLM p-adj=0.011), explaining 0.45% of the variation observed; and DGST48 (GLM p-adj=0.025), explaining 0.37% of the variation. In the inbredpanel, the SNP assay was also significantly associated with the yieldtrait DGST 48 (GLM p-adj=0.049; MLM p=0.017), explaining ˜1% of thevariation (R2=GLM 1.2%; MLM 0.9%). The SNP assay is located onchromosome 5.

SEQ ID NO: 25:

Three SNP assays were included in the analysis. In the inbred platformthe SNP assay was significantly associated with the yield trait DGST 24(GLM p-adj=0.001) explained 0.62% of the variation observed. In theinbred panel, the SNP assay was significantly associated with the yieldtrait DGST 24 (MLM p=0.015), explaining 0.83% of the variation. The SNPassay is located on chromosome 7.

SEQ ID NO: 28:

There were three SNP assays included in the analysis. In the inbredplatform, one SNP assay was significantly associated with the yieldtraits DGST 48 (GLM p-adj=0.028), explaining 0.37% of the observedvariation; and Oil (GLM p-adj=0.001), explaining 0.52% of the variation.The SNP is located on chromosome 1. There are two overlapping NSSmetaQTLs for the yield traits Starch and DGST 72.

SEQ ID NO: 31:

Three SNP assays were included in the analysis. In the inbred platform,one SNP assay was significantly associated with the yield traits DGST 48(GLM p-adj=0.001), explaining 0.5% of the variation observed; and DGST72 (GLM p-adj=0.022), explaining 0.4% of the variation. The SNP islocated on chromosome 2.

SEQ ID NO: 34:

Four SNP assays were included in the analysis. In the inbred platform,one SNP assay was significantly associated with the yield trait DGST 24:(GLM p-adj=0.001) explaining 0.6% of the variation observed. The SNP islocated on chromosome 10. There is an overlapping NSS metaQTL for theyield trait Starch.

SEQ ID NO: 37:

Four SNP assays were included in the analysis. In the inbred panel, oneSNP assay was significantly associated with the yield traits Starch (GLMp-adj=0.007; MLM p=0.0025), explaining ˜1.5% of the observed variation(R2=GLM 1.6%; MLM 1.5%); and DGST 72 (GLM p-adj=0.028; MLM p=0.0081),explaining ˜1.2% of the variation (R2=GLM 1.2%; MLM 1.2%). The SNP islocated on chromosome 8.

SEQ ID NO: 40:

Five SNP assays were included in the analysis. In the inbred panel, oneSNP was significantly associated with the yield traits DGST 24 (GLMp-adj=0.039; MLM p=0.019), explaining ˜0.9% of the variation (R2=GLM 1%,MLM 0.8%); and Protein (GLM p-adj=0.004; MLM p=0.005), explaining ˜1.3%of the observed variation (R2=GLM 1.4%; MLM 1.2%). The SNP marker islocated on chromosome 1. There is an overlapping NSS metaMTL for theyield trait DGST 72.

SEQ ID NO: 43:

There was one SNP assay included in the analysis. In the inbred panel,this SNP was significantly associated with the yield traits DGST 48 (GLMp-adj=0.007; MLM p=3.4×10⁻³), explaining ˜1.5% of the observed variation(R2=GLM 1.6%; MLM 1.3%); and DGST 72 (GLM p-adj=0.024; MLM p=4.3×10⁻³),explaining ˜1.3% of the variation (R2=GLM 1.4%; MLM 1.3%). The SNP islocated on chromosome 5.

SEQ ID NO: 46:

Four SNP assays were included in the analysis. In the inbred panel, oneSNP was significantly associated with the yield traits DGST 24 (GLMp-adj=0.001; MLM p=7.3×10⁻⁴), explaining ˜1.83% of the variationobserved (R2=GLM 2.1%; MLM 1.6%); and DGST 48 (GLM p-adj=0.003; MLMp=2.3×10⁻³), explaining ˜1.5% of the variation (R2=GLM 1.6%; MLM 1.5%).The SNP is located on chromosome 1. There is an overlapping NSS metaQTLfor the trait DGST 72.

SEQ ID NO: 49:

One SNP assay was included in the analysis. In the inbred panel, the SNPwas significantly associated with the yield traits Protein (GLMp-adj=9.9×10⁻⁴; MLM p=8.2×10⁻⁴), explaining ˜1.8% of the variationobserved (R2=GLM 1.8; MLM 1.7); DGST 48 (GLM p-adj=9.9×10⁻⁴; MLMp=1.2×10⁻⁴), explaining ˜2.4% of the variation (R2=GLM 2.5%; MLM 2.3%);and DGST 72 (GLM p-adj=9.99×10⁻⁴; GLM 3.8×10⁻⁴), explaining ˜2% of thevariation (R2=GLM 2%, MLM 2%). The SNP marker is located on chromosome5. This locus is in the vicinity of the loci associated with SEQ ID NOs:16 and 1, also associated with ethanol-related traits. There are NAMQTLs in this region of chromosome 5 for the yield traits Starch andProtein.

SEQ ID NO: 52:

One SNP assay was included in the analysis. In the inbred panel, the SNPwas significantly associated with the yield traits Protein (GLMp-adj=9.9×10⁻⁴; MLM p=2.8×10⁻⁴), explaining ˜2.1% of the variation(R2=GLM 2.2%; MLM 2%); DGST 48 (GLM p-adj=0.034; MLM p=5.4×10⁻⁴);explaining ˜1.7% of the variation (R2=GLM 1.4%; MLM 1.9%), and DGST 72(GLM p-adj=9.9×10⁻⁴; MLM p=1×10⁻⁵), explaining ˜3.2% of the variation(R2=GLM 3.3%; MLM 3.1%). The SNP marker is located on chromosome 10.There is a NAM QTL in this region of chromosome 10 for the yield traitStarch.

SEQ ID NO: 55:

In the inbred panel, the SNP was significantly associated with the yieldtrait Oil. The SNP marker is located on chromosome 1.

SEQ ID NO: 58:

Two SNP assays were included in the analysis. In the inbred platform,one SNP assay was significantly associated with the yield trait Protein:(GLM p-adj=0.017) explaining 0.5% of the observed variation. The SNP islocated on chromosome 4 (position 69.52 cM).

SEQ ID NO: 61:

Two SNP assays were included in the analysis. In the inbred panel, oneSNP assay was significantly associated with the yield traits Protein(GLM p-adj=0.028; MLM p=0.017), explaining ˜1% of the variation observed(R2=GLM 1.2%; MLM 0.9%); and Starch (GLM p-adj=0.002; MLM p=0.0037),explaining ˜1.6% of the variation (R2=GLM 1.7%; MLM 1.4%). The SNP islocated on chromosome 6.

SEQ ID NO: 64:

Two SNPs were included in the analysis. In the inbred panel, one SNPassay was significantly associated with the yield traits Protein (GLMp-adj=0.01), explaining 0.5% of the variation observed; and Oil (GLMp-adj=0.034), explaining 0.4% of the variation. The SNP is located onchromosome 7. This locus is close to the locus associated with SEQ IDNO: 19 that was significantly associated with the yield traits Protein,DGST 24, and Oil.

The SNPs disclosed herein were consistent in the General Linear Models(GLM) and in the Mixed Linear Models (MLM) discussed hereinabove. Assuch, the presently disclosed subject matter provides evidence thatthese SNPs, and markers comprising the same, can be employed forintrogression of yield traits from and into various Zea mays geneticbackgrounds.

REFERENCES

All references listed below, as well as all references cited in theinstant disclosure, including but not limited to all patents, patentapplications and publications thereof, scientific journal articles, anddatabase entries (e.g., GENBANK® database entries and all annotationsavailable therein) are incorporated herein by reference in theirentireties to the extent that they supplement, explain, provide abackground for, or teach methodology, techniques, and/or compositionsemployed herein.

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It will be understood that various details of the presently disclosedsubject matter may be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

What is claimed is:
 1. A method of producing a maize plant havingimproved digestibility or increased starch, the method comprising thesteps of: (a) isolating at least one nucleic acid from a population ofmaize plants; (b) genotyping said nucleic acid for the presence of amarker located within a chromosomal interval, wherein said chromosomeinterval is defined by and comprises SEQ ID NO: 37 and, wherein saidchromosome interval further comprises a C allele corresponding toposition 389 of SEQ ID NO: 37; (c) selecting a first maize plant on thebasis of the presence of the marker detected in (b); (d) crossing thefirst maize plant of (c) with a second maize plant not having saidchromosome interval; (e) collecting seed from the cross of (d); and (f)growing a progeny maize plant from the seed of (e), wherein said progenymaize plant comprises in its genome said chromosome interval, therebyproducing a plant with improved digestibility or increased starch. 2.The method of claim 1, wherein the maize progeny plant of (f) is ahybrid maize plant.
 3. The method of claim 1, wherein the first orsecond maize plant is a inbred.
 4. The method of claim 1, wherein thechromosome interval is located on Zea mays chromosome
 8. 5. The methodof claim 1, wherein the progeny maize plant is an elite maize plant. 6.The method of claim 1, wherein the maize progeny plant displays improveddigestibility at any one of 24 hours, 48 hours or 72 hours as comparedto a control plant.
 7. The method of claim 1, wherein the chromosomeinterval is detected through the use of a PCR primer pair that annealsto any one of SEQ ID NOs: 37 or 123, wherein the primer pair is capableof initiating DNA polymerization by a DNA polymerase on a Zea maysnucleic acid template to generate a amplicon diagnostic for the presenceof said chromosome interval.
 8. The method of claim 7, wherein the PCRprimer pair comprises SEQ ID NOS: 38 and
 39. 9. The method of claim 1,wherein the maize progeny plant further comprises at least one otherlocus in its genome selected from the group consisting of MTL1, MTL2,MTL3, MTL4, MTL5, MTL7, MTL8, MTL9, MTL10, MTL11, MTL12, MTL13, MTL14,MTL15, MTL16, MTL17; and MTL18.